workshop 11 a and b lymphokines

Workshop 11 A and B Lymphokines

GALE GRANGER and DAVID WOOD

Harvard Medical School and Massachusetts General Hospital, Boston, Massachusetts, USA

Interleukin-l production by a human monocyte cell line is induced by a T lymphocyte product

E. P. AMENTO, J. T. KURNICK, S. M. KRANE

The human monocyte cell line, U937, displays monocyte/macrophage characteristics upon exposure to conditioned medium from lectin-stimulated peripheral blood or cloned T lymphocytes or the medium from a mixed lymphocyte reaction. We recently reported (Clin. Res. 30, 543A 1982; PNAS, in press) that similarly stimulated U937 cells produced a monokine, mononuclear cell factor (MCF), which increased prostaglandin E2 and collagenase production by cultured rheumatoid synovial cells. We previously showed that MCF had lymphocyte activating factor (LAF) activity and conversely that LAF was active in a MCF assay. U937 cells, however, have never been reported to produce LAF, and, indeed, in our own hands the crude conditioned medium from lymphokine stimulated U937 cells lacked LAF activity. These disparate observations were the subject of our experiments. Cloned human T lymphocytes were washed free of their growth supporting medium and stimulated with lectin. U937 cells were stimulated with the cell-free supernate from the lectin-stimulated T lymphocytes and the conditioned medium chromatographed on an AcA54 column. MCF and LAF activities were co-eluted in the range of Mr 15-20,000 suggesting the presence of an inhibitory substance in the unfractionated medium. Indeed, fractions of approximately Mr 45-60,000 were inhibitory to T lymphocyte activation of both thymus and spleen origin, but did not prevent MCF activity. Thus, U937 cells in the presence of a lymphokine from lectin-stimulated cloned human T lymphocytes produce IL-l as assayed by both MCF and LAF activities. The previous inability to demonstrate IL-l activity from U937 cells is due to an inhibitor of lymphocyte proliferation which masks the LAF assay. Utilizing cloned T lymphocytes and a monocyte cell line we can demonstrate the influence of T lymphocyte factors on monocyte maturation and monokine production. These data suggest that it is the antigen-specific cell (i.e. the T lymphocyte) which is responsible for initiating the immune response via activation of the monocyte/macrophage.

Jefferson Medical College, Philadelphia, PA 19107, USA

Ontogeny of IL-2 responsive cells in the fetal thymus

M. H. BOCCHIERI, O . BATUMAN, S. HAUPTMAN, and J. B. SMITH

We have examined the ontogeny of IL-2 responsive cells in the fetal thymus of CBA mice at 14, 16 and 18 days gestation. The responses of these thymocytes to Con A alone, T cell growth

15th International Leucocyte Culture Conference, Asilomar' 277

factor (TCGF) alone, purified IL-2 alone, and to co-stimulation by Con A plus TCGF or purified IL-2 were measured and compared to those of neonatal and adult thymocytes. The peak proliferative response of 1 X 105 thymocytes to co-stimulation was found after four days incubation. An adult-level TCGF/IL-2 alone response was found in the neonate, although 18 day fetal cells did give a response of lower magnitude. The co-stimulator response was detectable as early as 16 days gestation and achieved adult levels by 18 days. The background proliferation of thymocytes in medium alone was significantly greater in cultures of 14 day fetal thymocytes than at any other age tested.

Biological Research and Therapy Branch, NCI-FCRF, Frederick, MD 21701, and Developmental and Metabolic Neurology Branch, NINCDS, Bethesda, MD 20205, USA

Effect of interferon (IFN) on cholesterol content of human peripheral blood mononuclear cells

P. BOUGNOUX, N. SALEM, E. BONVINI, H. C. STEVENSON, and T. HOFFMAN

Interferon has been shown to alter membrane fluidity. Since changes in cholesterol content are known to be important determinants of membrane viscosity, we have investigated the IFN-induced changes in 3H-cholesterol incorporation in human peripheral blood cells. Monocytes were purified by countercurrent centrifugal elutriation and pretreated for one hour with a-(recombinant) IFN (1000 U/ml), then incubated with 3H-cholesterol in RPMI-1640 medium containing 1 % fetal bovine serum. The lipid extract was separated by thin layer chromatography and the radioactivity migrating with the same ~ value as the internal cholesterol standard was measured. Monocytes, pretreated with IFN (1000 U/ml) incorporated 2367 ± 43 cpm compared with medium-treated monocytes which incorporated 3008 ± 87 cpm. We also determined the cholesterol content of lymphocytes by a colorimetric method after their treatment with either a-(recombinant) or 13-(fibroblast) IFN (1000 U/ml) for 18 hours. The molar ratio of cholesterolilipid phosphorus was 0.37 for medium-treated and 0.32 and 0.31 for a- and 13-IFN treated cells, respectively. These data suggest that decreased cholesterol content may be one of the molecular bases for changes in the physical state of the membrane consequent to IFN treatment.

Walter Reed Army Medical Center, Washington DC, USA

A neutrophil-activating factor from a continuous lymphoid cell line mediates increased number of neutrophil C3b surface receptors and increased functional activity

A. CROSS, M. BERGER, G. LOWELL, and J. SADOFF

We have previously described a neutrophil activating factor (NAF) of approximately 30,000 MW from supernatants of a continuously cultured human B Iymphoblastoid (Raji) cell line (Clin. Res. 29: 382A, 1981). This NAF enhances neutrophil chemokinesis but is. not a chemoattractant. This NAF requires preincubation of neutrophils as well as the presence of exogenous gelatin for optimal expression of the enhanced chemokinetic activity by neutrophils. We now report that NAF-treated neutrophils (NAF-N) generated nearly Ph times

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more superoxide as measured by superoxide dismutase-inhibitable ferricytochrome C reduction after stimulation with phorbol myristic acetate (PMA) than control-treated neutrophils (C-N). After 3 hours pre-incubation but before PMA stimulation, NAF-N had 2% times more hexose monophosphate shunt activity than C-N, and this difference was also observed after stimulation with PMA. In a slide phagocytic (Cytospin) assay a significantly higher percentage NAF-N cells were infected with E. coli than C-N and each NAF-N had significantly more bacteria associated with it. This was true when neutrophils were mixed with bacteria in the complete absence of opsonins and when neutrophils were mixed with bacteria in the presence of heat-inactivated serum. Finally, NAF-N had markedly increased ability to form rosettes with sheep EA(IgM)-C3b but showed no difference in ability to rosette with EA(IgG). This was more demonstrable with small amounts of C3b coating the red blood cells. This implies a specific increase in the neutrophil C3b receptor activity without a similar change in the Fc receptor activity. We conclude that NAF activates neutrophils in vitro and induces C3b surface receptors. These increased C3b receptors, or perhaps other neutrophil surface receptors, and increased metabolic activity may then mediate the observed increase in neutrophil function in vivo. NAF may thus be an important mediator between mononuclear cells and neutrophils in inflammatory reactions.

INSERM U 131, 32 rue des Carnets, Clamart, France

Characterization of B cell derived immunoregulatory product. Possible role in the modulation of the antibody production

P. DEL GUERCIO

The supernatant from non antigen and non mitogen-stimulated spleen cells is effective in augmenting the IgM antibody response to SRBC or DNP-HRBC, whether added to cultures on day O. This regulatory activity was found to be mediated via a soluble product which is released by B lymphocytes. A product which exhibits the activities ascribed to the factor produced by normal B lymphocytes was also produced by a B cell clone that does not express immunoglobulin heavy or light chains. The biologic activities of this product can be distinguished from those attributed to IL-2, produced by T cell line lymphocytes, since 1. it does not promote the proliferation of Con A-treated T cells, 2. it does not provide T cell help for the generation of PFC response to heterologous erythrocytes. The B product enhances the antibody immune response by preventing the activation of T suppressor cells. Cloned B cells provided a source of the factor that facilitated its characterization. The M. W. was measured by gel filtration. The sensitivity to enzymatic or chemical treattnent was established.

University of Iowa and V. A. Hospital, Iowa City, Iowa, USA

L ymphokine secretion by lymphocyte subpopulations in the rat

T. L. FELDBUSH, D. M. LUBAROFF, K. THOMAS, H. D. HUNT, and C. LYNBERG

Rat lymph node cells have been separated into subpopulations using antibody-coated Degalan bead columns. Surface immunoglobulin (sIg) positive (B) cells and sIg negative (T)


cells were obtained as adherent and non-adherent populations from a column coated with F(ab')z-specific rabbit anti-rat immunoglobulin. All the sIg-negative cells possess the rat alloantigen ART -1 whereas only 50-60 % of these T cells carry the ART -2 peripheral T cell alloantigen. Further separation of the Ig-negative cells was accomplished using antisera to ART-2. T cells were incubated with anti-ART-2 serum and separated on a Degalan bead column coated with Fc-specific anti-rat Ig serum into ART -2-enriched (adherent) and ART -2-depleted (non-adherent) populations. Our data thus far would indicate that, as anticipated, only T cells are active in the production of Interleukin-2 (IL-2). In addition, both ART-2-enriched and depleted cell populations have been shown to secrete this lymphokine, although the cells depleted of ART-2-bearing T lymphocytes may be more reactive than the cells enriched for the ART-2 lymphocytes. The production of additionallymphokines, including Interleukin-3 (IL-3), T-Cell Replacing Factor (TRF), B-Cell Growth Factor (BCGF), and BCell Stimulating Factor (BCSF) are also being studied using separated rat lymphocytes.

Cleveland Clinic Foundation, Cleveland, OH, USA

Estrogen stimulated production of IL-l from human placental derived macrophages

A. FLYNN, J. H. FINKE, and M. L. HILFIKER

Human placentas have been described to contain numerous macrophage-like cells. We have studied the ability of these phagocytic cells to produce Interleukin-1 (IL-1) and their hormonal stimulation by estrogen. Macrophages were isolated enzymatically and by plastic adherence from post-delivery normal human placentas (we have been able to isolate by these simple techniques greater than 1011 cells from a single placenta). In the thymocyte proliferation (TP) assay and an Interleukin-2 (IL-2) assay using a long-term IL-2 dependent T cell line, these cells in 48 hour culture were shown to be producing IL-1, but not IL-2 without any in vitro stimulation. Gel filtration of 48 hour culture supernatant fluids indicated that the greatest TP activity was found in fractions containing proteins of about 17,000 daltons. Phagocytosis of latex beads for 4 hours also resulted in greater release of IL-1 from the placental adherent cells. We subsequently studied the stimulation of these cells by hormones to determine if activation could occur by this means in vitro and perhaps in vivo. Initially, we studied the ability of estradiol and progesterone to stimulate IL-1 production in the macrophage-like tumor cell line P388D1• A dose response for estrogen activation of macrophages to produce IL-1 was calculated between 1 X 10-7M and 1 X 10-5M estradiol. Estradiol was found at a dose as low as 1 X 10-6M to stimulate IL-1 production and release by the P388D1 cells. At higher doses estradiol was more active than the positive control, phorbol myristate acetate (PMA) (1 I-Ig/ ml), in stimulating IL-l production. Progesterone at levels similar to estradiol did not activate the P388D1 cells to produce any measurable IL-l. Estradiol stimulation of placental derived macrophages likewise resulted in significant production/release of IL-l. Estrogen stimulated placental macrophages are an abundant source of human IL-1 for further characterization of this immune factor. Further studies on a relationship between estrogen and placental macrophages may also explain physiological responses in pregnancy that may relate to IL-1 activity.


University of Minnesota, St. Paul, MN 55108, USA

Characterization of helper factors distinct from interleukin-2 necessary for the generation of allospecific cytolytic T lymphocytes

R. D. GARMAN and D. P. FAN

The in virro differentiation of primary allospecific cytolytic T lymphocytes (CTL) from BALB/c (H-2d) spleen cell precursors in response to irradiated RDM4 (H-2k) tumor cells will not occur unless the cultures are supplemented with exogenous helper factors. Such factors can be provided by conditioned medium (CM) from cultures in which Sendai virus-immune BALB/c spleen cells are stimulated either with Sendai-infected cells (SC-CM) or with peptides cleaved by CNBr from intact virions (SP-CM). To determine the relationship of these CTL helper factors (CHF) to the known cytokines interleukin-l (IL-l), interleukin-2 (IL-2) and immune interferon (y-IFN), kinetic and biochemical experiments were carried out. CHF activity in both SC-CM and SP-CM peaked after day 3. In contrast, IL-2 activity, determined from the proliferation of the IL-2 dependent cell line CTLL-20, peaked at day 2 and dropped to background levels after day 3. Fractionation of day 4 SC-CM by gel filtration revealed peaks of CHF activity in the void volume and in a single peak at an apparent molecular weight of 17,000 (CHFI7). A small amount of IL-2 (0.06 U/ml) eluted with the typical apparent molecular weight of 30,000 and contained no CHF activity. In contrast, fractionation of day 4 SP-CM by gel filtration showed a single peak of CHF activity at an apparent molecular weight of 30,000 (CHF30). A small amount of IL-2 (0.03 U /m!) eluted in the same region. From results of hydrophobic chromatography of CHF17 and CHF30 on phenyl-Sepharose, the majority of CHF activity is more hydrophilic than IL-l, IL-2 and y-IFN. (HILFIKER et a!. 1981. J. Immuno!. 127: 1983; OPPENHEIM et al. 1982. Fed. Proc. 41: 257.) Taken together, these results suggest that the inability to generate a strong allospecific CTL response to irradiated tumor cells in this system is not due to a lack of IL-l, IL-2 or y-IFN, but to a lack of additional CHF. This helper activity can be provided by at least two molecules with different apparent molecular weights (17,000 and 30,000). Studies are in progress to determine the biochemical relationship of CHF17 to CHF30.

Institute of Immunology, University of Heidelberg, and "Instirute of Immunology and Genetics, German Cancer Research Center, Heidelberg, FRG

Molecular and functional heterogeneity of two T cell derived macrophage activating factors

D. GEMSA, K.-M. DEBATIN, C. KUBELKA, U. KEES<-, and P. H. KRAMMER"

Macrophages are induced to an enhanced metabolic and functional activity by macrophage activating factor (MAF). MAF may be a heterogeneous mixrure of different factors. To analyze this problem, MAF-cont:ilning supernatants of two Concanavalin A-stimulated long-term T cell clones, PC-AKR-96 (96) and PK-AKR anti-B6 7.1.2 E8 (7.1.2 E8), were tested on resident peritoneal macrophages from DBA/2 mice. Within 24 hours both MAFs stimulated glucose consumption via the hexose monophosphat shunt, incorporation of 14C-glucosamine, and Oi

15th International Leucocyte Culture Conference, Asilomar· 281

and HzC:)z release. In contrast, RNA synthesis as determined by jH-uridine incorporation into acid-precipitable material was rapidly enhanced with MAF 7.1.2 E8 and strongly reduced with MAF 96 after 24 hours of incubation. Protein synthesis was stimulated by MAF 7.1.2 E8 and slightly diminished by MAF 96 after 50 hours of incubation. Release of prostaglandin Ez, associated with activation of macrophages to suppressor cells, was highly elevated with MAF 96 but unaffected by MAF 7.1.2 E8. Only MAF 7.1.2 E8 was capable of stimulating pinocytic and phagocytic activities whereas MAF 96 was able to activate macrophages to kill schistosomula. MAF 96 and MAF 7.1.2 E8 at a concentration of 0.05 % and 2 %, respectively, were similarly able to activate resident macrophages to a high tumor cytostatic activity against Eb lymphoma cells. However, only MAF 96 but not MAF 7.1.2 E8 was capable of inducing tumor cytolytic macrophages. Qualitative differences between both MAFs could be further substantiated by the use of a rabbit anti-lymphokine antiserum in that only the MAF 96-dependent activation could be abrogated. The findings indicate that there are at least two distinct types of MAF that share the property to stimulate glucose metabolism, release of oxygen metabolites, glucosamine incorporation and induction of tumor cytostatic macrophages. They differ, however, in their capacity to stimulate RNA, protein and arachidonic acid metabolism, and to induce of schistosomulicidal and tumor cytolytic macrophages.

Laboratory of Immunodiagnosis, NCI; Tissue Bank, Naval Medical Research Institute; +1nstitute for Immunology, Erlangen, FRG; *Institute for Immunology, Bern, Switzerland

Characterization of cell lines enhancing interleukin-2 production of PHA-stained human T lymphocytes

M. GRAMATZKI+, D. M. STRONG, G. D. BaNNARD", and R. B. HERBERMAN

1nterleukin-2 (IL-2) has been shown to play an important role in cellular immune interactions. Previously, it was found that the addition of small numbers of a B lymphoblastoid cell line could strongly enhance 1L-2 production of phytohemagglutinin (PHA) stimulated human lymphocytes (BONNARD et aI., Cell. Immunol. 51: 390, 1980). To study the mechanisms involved in this effect, a large variety of mycoplasma-free human cell lines (CL) was tested for their IL-2 enhancing capacity. It was found that the CTL had to be present during production, because supernates of PHA-treated or untreated CL were ineffective. All B-lymphoblastoid CL tested enhanced, even when they were immature, surface immunoglobulin-negative (EW36, NALM-1) negative for EBV-associated antigens or Fe-receptors (BJAB), without HLA-A,B,C antigens (Daudi) or had like the HLA-DR deficient mutant 6.1.6 (donated by Dr. D. Pious) no detectable DR determinants. Interestingly, autologous as well as allogeneic B-CL provided accessory function for IL-2 production. In contrast, the macrophage line U-937 and DR-negative T-CL from patients with acute lymphoblastic leukemia as well as the nonlymphoid lung cancer CL ChaGo showed no enhancement. However, the DR-positive T-CL established from mature cutaneous T-cell lymphoma (HUT78 , HUT102) enhanced IL-2 production. Extensive surface marker analysis revealed no obvious common characteristic of the enhancing CL. Although DR specificities could be ruled out as an important factor, the finding that all of the DR positive CL independent of their lineage could enhance, suggests that the reaction may be related to some product of the major histocompatibility complex. The enhancing determinant appears to serve as an important additional signal provided by the CL to the PHA-activated 1L-2 produce cell.

Supported in part by NCI Contract Y01-CB-00319 & DFG Gr689-1.


Minerva Institute for Medical Research, PB 819, SF-00101 Helsinki 10, Finland

Leukocyte migration inhibition effect of angiotensin-I-converting enzyme

C. GRONHAGEN-RISKA, F. FYHRQUIST, I. PETTERSSON, M.-G. WELIN, and T. WEBER

Angiotensin-I-converting enzyme (ACE) is a membrane-bound and circulating glycoprotein produced by vascular endothelial cells. Its two known functions are to convert angiotensin (A) I into vasoactive A II and to inactivate bradykinin. Elevated serum ACE activity has been observed in sarcoidosis and in a number of other macrophage engaging diseases, such as leprosy and primary biliary cirrhosis. There is firm evidence, that macrophages may produce this enzyme, and cells from sarcoid granulomas have been shown to contain ACE. The reason for this is not known, but certain immunological functions for ACE have been suggested (1, 2). We have purified human lung ACE and added it, 25-20 !Jog/mI, into a direct leucocyte migration inhibition factor (UF) assay (3). ACE caused up to 50 % migration inhibition as compared to controls. The cell response was dose-dependent and not due to any toxic effect. ACE caused similar responses in neutrophils separated from mononuclear cells as in unseparated cell suspensions. In a separate experiment ACE did not activate peripheral blood lymphocytes in vitro. Migration inhibition was not affected by the specific ACE inhibitors captopril or MK-421. These in vitro experiments suggest, that ACE contributes to the cellular reactions associated with macrophage engaging diseases. This effect is tentatively not associated with the known enzymatic activity of ACE.

1. WEINSTOCK, J. V., M. N . EHRINPREIS, D. L. BOROS, and ]. B. GEE. 1980. Effect of SQ 14225, an inhibitor of angiotensin I-converting enzyme, on the granulomatous response to Schistosoma mansoni eggs in mice. J. Clin. Invest. 67: 931.

2. SCHRIER, D. J., L. M. RIPANI, A.-L. KATZENSTEIN, and V. L. MOORE. 1982. Role of angiotensin-converting enzyme in Bacille Calmette-Guerin-induced granulomatous inflammation. J. Clin. Invest. 69: 651.

3. WELIN, M.-G., T. MAKINEN, and T. H. WEBER. 1982. Leucocyte migration inhibition assay from clotted plasma droplets. J. Clin. Lab. Immunol. 8: 65.

Department of Virology and the Laboratory for Cell and Tissue Culture Research, University of Umea, S-901 87 Umea, Sweden

Normal and interferon(IFN)-induced pattern of protein synthesis in human large granular lymphocytes (LGL), T cells and monocytes

A. GUSTAFSSON, T. Ny, and E. LUNDGREN

LGL, T cells and monocytes from the same donors were prepared from human buffy coats by centrifugation on Percoll gradients as described (1). The preparations were characterized with regard to antigenic expression (as determined by reactivity with OKT3, OKTIO and OKMI monoclonal antibodies), morphology and natural killer (NK) cell activity. LGL were over 80 % pure, a majority of the cells expressed the OKT3-/0KTlO+/OKMl + phenotype and the cells were highly enriched for NK cell activity. T cells were non-cytotoxic and over


95 % of the cells were characterized as OKT3 + IOKTIO- IOKMI- . Monocytes contained over 95 % OKT3 - IOKTI0- IOKMI + cells. The pattern of protein synthesis in these cells was determined by labelling the cells with 35S-methionine and separating cell extracts by twodimensional electrophoresis. Over 90 % of the synthesized proteins were similar in LGL and T cells, whereas several differences (also in the expression of major proteins) existed between these two cell types and the monocytes. Treatment of LGL, T cells and monocytes with IFN lead to the rapid increase in synthesis rate of eight specific proteins. These proteins were expressed to the same degree in all three cell preparations, despite the marked differences in cell phenotype. These data suggest that 1. LGL are more closely related to T cells than to monocytes, 2. the augmentation of NK cell activity by IFN is not mediated through the increased synthesis of LGL-specific proteins, but might be due to interactions between generally occurring, IFN-inducible proteins and preexisting, LGL-specific (lytic) proteins.

1. ORTALDO, J. R., S. O. SHARROW, T. TIMONEN, and R. B. H ERBERMAN. 1981. J. Immuno!. 127: 2401.

Department of Pathology, Georgetown University, Washington, D.C., USA

Asbestos-related in vivo fibrogenesis, induced by lymphokines

D. P. HARTMANN, N . A. RAO, and E. KAGAl'i

Asbestosis is a diffuse interstitial pulmonary fibrosis. Recent evidence indicates that immunologic mechanisms may playa role in the pathogenesis of asbestosis (1). In an earlier study, alveolar macrophages from asbestos-exposed rats were known to trigger autologous splenic lymphocyte proliferation. It is conceivable that immunologic mediators (macrokinesl lymphokines) are responsible for this phenomenon and for pulmonary fibrosis. In order to further elucidate the mechanisms involved, we have assayed macrophage-lymphocyte coculture supernatants for the presence of interleukin-l (IL-l), interleukin-2 (IL-2) and for in vivo fibrogenic activity. Alveolar macrophages and autologous splenic lymphocytes were obtained from groups of asbestos-exposed and sham-exposed Fischer 344 rats. Co-cultures were maintained at 37 °C, in 5 % CO2 for 48 hr. Thereafter, supernatants were collected and tested for IL-l (using C3H/HeJ mouse thymocyte proliferation assay) and IL-2 activity (using CT-6 cytotoxic T cell proliferation assays, in Dr. J. J. Farrar's laboratory, NIDR). Fibrogenic activity was evaluated in vivo by the injection of 0.2 ml of supernatants into the vitreous cavity of Dutch pigmented rabbits . Supernatants from asbestos-exposed rats, that were subsequently immunized with antigen, were found to contain both IL-l and IL-2 activity. On the other hand, supernatants from immunized, sham-exposed rats contained lower levels of both IL-l and IL-2. A pre-retinal membrane was observed within 4 days of intra-ocular injection of supernatants from asbestos-exposed rats. Studies are at present being conducted to investigate the makeup of the membrane.

In view or recent evidence indicating that IL-l and other thymocyte activating factors are capable of influencing several in vitro fibroblast functions and may be important in modulating inflammatory responses, our findings may have important pathogenetic relevance to the host response to inhaled asbestos.

1. KAGAN, E. 1981. The alveolar macrophage: Immune derangement and asbestos-related malignancy. Sem. Oncol. 8: 258.


Department of Anatomy, University of Nebraska Medical Center, 42nd and Dewey Avenue, Omaha, Nebraska 68105, USA

Dioxin may suppress immune responses by inhibiting lymphokine production

J. D. JACKSON, D. A. CROUSE, and J. G. SHARP

Dioxin (2,3,7,8-tetrachlorodibenzo-p-dioxin or TCDD) is a toxic chemical which occurs as a contaminant in the production of some chlorinated phenols and herbicides. It has been implicated as a carcinogen and teratogen in mice and rats with potentially the same effects in man. When 5 Jlg/kg dioxin is administered to C57B1/6 female mice per os, significant suppression of DTH, hemolytic plaque formation, and lymphocyte mitogenic responses to PHA, Con A and LPS is observed for single and multiple dose protocols. However, it has no effect on either cell mediated T cell cytotoxicity against P815 targets or on NK cytotoxicity against YACI targets. These data suggested that the target of dioxin mediated immunosuppression might be the helper T cell. Consequently, we investigated the relative distribution of total, helper, cytotoxic and suppressor T cells on the basis of surface marker phenotype in the thymus and spleens of untreated controls, vehicle treated controls (corn oil and acetone), and dioxin treated mice. We employed monoclonal reagents in a biotin-avidin system and the Ortho SOH cytofluorograph to detect Thy 1 +, Lyt 1 +, Lyt 2+ and SMlg+ cells. Background staining by fluorescinated avidin was corrected in channel by channel subtraction. (Note: In these studies we report only bright Lytl + cells.) The relative distribution of cells in the spleen was - 32 % Thy 1 +, - 32 % Lytl +, - 15 % Lyt2 + and - 28 % SMIg+ in all groups (dioxin treated, vehicle treated and control). In the thymus where dramatic weight changes occur following treatment, the surface marker phenotype profiles are again not changed significantly from normal C95 % Thy! +, - 46 % Lytl +, - 71 % Lyt2+). Thus the relative distribution of helper T cells was unchanged by dioxin treatment and the simplest explanation of these observations is that dioxin suppresses immune responses by inhibiting Iymphokine (or monokine) production.

Supported by UNMC Seed Funds.

UCLA, School of Medicine, Los Angeles, Ca. 90024, USA

Production of human interleukin-2 with minimal contaminants

V. KERMANI-ARAB, V. A. DERBALIAN, S. A. PATEL, and J. L. FAHEY

Kinetic experiments were conducted to obtain optimum condition for high yield production of Interleukin-2 (IL-2) without complicating substances such as serum components, 12-0-tetradecanoyl-phorbol-13 acetate (TP A), or B cell lines. Normal human peripheral blood mononuclear cells at several concentrations (1 X 106 to 9 X l~/ml) with or without adherent cells were cultured in RPMI-1640 containing 1 to 50 J.tg Phytohemagglutinin P (PHAp)/ml for 24 to 72 hours at 3rC, 5 % Co2, The PHA was removed from the cultures at different intervals (0 to 8 hours) after initial incubation, and then the cells were reincubated to complete the incubation period. IL-2 activity was determined by CTLL-2 assay. Lymphocyte exposure to PHA for at least 4 hours was needed for substantial IL-2 production. Maximum IL-2 production was seen after 48 hours of cultures. IL-2 produced in serum free/PHA free cultures (after terminating brief PHA exposure) demonstrated 30 fold greater specific activity


(Units IL-2) P . when compared to corresponding cultures containing PHA + B cell line

mg rotem + 0.25 % BSA. This technique provides an economical product that is useful for further purification and biochemical studies of IL-2, and for investigation of IL-2 responsive lymphoid cells systems.

Supported in part by NIH Grant No. CA-12800.

!Institut fur Immunologie und Genetik, 2Institut fur Virologie, Deutsches Krebsforschungszentrum, Heidelberg; 3Institut fur Immunologie und Serologie, Universitat Heidelberg, Heidelberg; 4Abteilung fur Exp. Himatologie der Gesellschaft fur Strahlen- und Umweltforschung, Munchen, FRG

Segregation of production of macrophage activating factor (MAF), colony stimulating factor (CSF) and immune interferon (IFN-y) in Tcell hybridomas derived from fusions with a selected high producer T cell clone in long term culture secreting all three lymphokines

P. H . KRAMMER!, B. KALTMANN', L. HULTNER\ U . KEESI, F. MARCUCCI2, and D. GEMSA3

Murine (AKR/J) T cell clones were selected for high production of lymphokines upon T cell mitogen (concanavalin A) stimulation. Some clones released MAF, CSF and IFN-y. MAF induced a variety of metabolic and functional parameters upon incubation with resident macrophages, most notably prostaglandin E2 release, schistosomula killing, and tumor cytolysis. CSF and various subtypes were detennined by their ability to induce the fonnation of colonies of mature haematopoietic cells in semi-solid agar cultures of bone marrow cells, and IFN-y was measured in a virus-plaque reduction assay. Quantitatively different levels of production of MAF, CSF, and IFN-y of different subclones suggested that the above Iymphokines are molecularly not identical. This assumption was strengthened by segregating Iymphokine release from hybridomas derived from fusions of such T cell clones with the AKR T cell tumor BW5147. These findings are discussed with respect to the possibility that lymphokines may belong to a multigene family, and that a particular T cell may be capable of releasing severallymphokines simultaneously. Assuming that some of these Iymphokines act on a variety of different target cells, Iymphokine activity must be stringently regulated.

Harvard Medical School and Massachusetts General Hospital, Boston, Mass., USA

Cloned human T lymphocytes produce a soluble factor which modulates a human monocyte cell line (U937)

J. T. KURNICK, E. P. AMENTO, and S. M. KRANE

Cloned human T lymphocytes produce a soluble factor which inhibits proliferation, while inducing maturation, of a human monocyte cell line (U937). While many studies have focussed on antigen-presentation by macrophages and interleukin-l (IL-l) induction of T lymphocyte proliferation via interleukin-2 (IL-2), the lymphocyte's role in macrophage activation must likewise be stressed. We have studied the soluble products of T lymphocytes activated either by specific soluble antigen (in the presence of compatible macrophages), or by lectin in the absence of macrophages. Soluble antigen-specific T lymphocytes stimulated with macrophages


and antigen produce IL-2 as well as a factor which inhibits U937 proliferation in a dosedependent fashion. Lectin-stimulated T lymphoblasts, in the absence of macrophages, also produce a factor which modulates U937, but such T cells produce no IL-2 activity. Column fractionation indicates that the T lymphocyte factor which is active on U937s is eluted in the range of 45--60,000 daltons. Clones of either OKT4 (helper) or OKT8 (cytotoxic) T lymphocytes can produce active factor. This same factor induces cell adherence and soluble products from the U937s including IL-1 (as demonstrated in a companion abstract). Proliferation of transformed human Band T lymphocyte cell lines are not affected by this same material, indicating that the factor is cell type specific. Thus, the cessation of growth of the U937s upon incubation with the T cell product appears to be a result of a maturation signal after which the U937s acquire characteristics of a more mature monocyte/macrophage. These findings indicate that a soluble T cell product (?Interleukin-O) is responsible for maturation and monokine production. The relationship between the T lymphocyte factor which is active on the U937 cell line and previously described factors which influence macrophage activity (such as migration inhibition factor) is under investigation.

Inst. of Microbiology, University of Torino, Italy, and DKFZ, 6900-Heidelberg, FRG

Immune (y) interferon production by murine T -cell lymphomas

S. LANDOLFO, B. ARNOLD, M. SARZOTII, and E. BALZI

Various cloned murine T-cell hybridomas and T-cell lymphomas were evaluated for their ability to produce IFN-y, macrophage activating factor (MAF) and interleukin-2 (IL-2). Two T cell clones L12-R1 and L12-R4 derived from a spontaneously in vitro transformed cell line L12 which was originally established from fetal calf serum-primed C57BLl6 spleen cells were found to produce IFN after mitogen stimulation. Phorbol myristate acetate (PMA) led to maximal IFN production « 2,000 IU) by L12-R4 cells at concentrations of 2 X 107M. None of the cell lines tested produced IFN either constitutively or upon lipopolysaccharide (LPS) stimulation. The IFN was characterized as immune (y) IFN being labile at pH 2 and neutralized by 2 rabbit anti-murine IFN-y antisera but not by antiserum to murine IFN-a and -~. L12-R4 IFN-y chromatography on Sephacryl 5-200, Phenyl Sepharose and Con ASepharose revealed a molecular weight of about 45,000 d, a significant apparent hydrophobicity and a glycidic moiety just as other murine IFN-y. In addition to IFN-y, IL-2 and MAF were also found in the supernatants of the mitogen treated L12-R4 cells. Our data provide further evidence that IFN-y is a product of T cells of Lyt-1 +, 23 - phenotype and establish a system which could be used for the IFN-y purification as well as for the characterization of the molecular mechanisms involved in T cell differentiation leading to IFN-y production.

Supported in part by CNR-PFCCN Grant 81.01368.96.

NIDR, NIH, Bethesda, MD 20205, USA

Heterogeneity of human epidermal cell derived thymocyte activating factor (ET AF)

T. A. LUGER, J. A. CHARON, and J. J. OPPENHEIM

Both normal epidermal cells and keratinocyte cell lines, devoid of Langerhans cells, produce mediators such as ETAF that augment immune responses. ETAF up to now is biologically and


biochemically indistinguishable from macrophage derived Interleukin-l (IL-I). ETAF stimulates a variety of target cells, including thymocytes, lymphocytes, fibroblasts, neutrophils, monocytes, and hepatocytes. It is, therefore, possible that different species of ET AF rather than one molecular species may be responsible for this multiplicity of biological activities. In support of this idea human ETAF exhibits molecular weight (m.w.) heterogeneity; ETAF eluted as 2 peaks off Sephadex S-200 at m.w. 12-20K and 40-70K. However, as is the case with IL-I, the high m.w. fortn of ETAF appears to be an aggregate and can be recovered in the low m.w. fortn upon rechromatography. In addition, isoelectrofocusing (IEF) as well as chromatofocusing of ET AF resulted in 3 isoelectric peaks of thymocyte growth enhancing activity at pI's of 7.2, 6.0, and 4.9. ETAF partially purified by S200 gel filtration was chemotactic both for polymorphonuclear leucocytes (PMN) and mononuclear leucocytes (MNL). After separation of ETAF by IEF into 3 different peaks of equivalent thymocyte enhancing activity, the chemotactic activity for PMN did dissociate from that for MNL. ETAF at pI 4.8 was most chemotactic for MNL but not PMN, whereas ETAF at pI 7.2 attracted predominantly PMN's but not MNL's. This represents the first evidence that the charge heterogeneity of ET AF is associated with different biological activities on distinct target cells. Thus, these biochemically separable moieties of ET AF, and possibly IL-I, may actually represent distinct mediators.

Institute of Microbiology and Immunology, School of Medicine and Laboratory for Cellular Immunology, Institute for Biological Research, Belgrade, Yugoslavia

Correlation between IL-2 activity in the spleen and susceptibility to the induction of EAE in rats

M. L. LUKIC, L. EJDUS-KoNSTANTINOVIC, and MARIJA MOSTARICA-STOJKOVIC

Albino Oxford (AO) and Dark August (DA) strains of rats exhibit different susceptibility to the induction of experimental allergic encephalomyelitis (EAE). AO rats being resistant to the induction of the disease with myelin basic protein (BP). Spleen cell culture supernatants, rich in IL-2 activity was found able to condition BP-sensitized lymphocytes to transfer EAE. Therefore we have studied whether cells of AO and DA rats differ in their production and responsiveness to interleukins. It was found that IL-2 activity was significantly higher in culture supernatants obtained from the Con-A stimulated cells of DA rats irrespective of whether they were tested by measuring growth production of DA or AO T cell blasts. IL-2 production in both strains was highly macrophage or II-I dependent. However, independently of the source of II-I preparation Il-2 activity was higher in the culture supernatants of splenic T cells obtained from DA rats. Thus, it appears that lower 11-2 activity correlate with the lower susceptibility to the induction of EAE. Resistance to the induction of EAE in AO rats was abrogated by the procedures which eliminate the precursors of suppressor T cells (1 ). Experiments in progress are aimed to show whether the observed lower susceptibility to the induction of T cell-mediated autoimmunity and lower B-2 activity in AO rats are the consequences of the similar suppressive mechanisms.

1. MOSTARICA-STOJKOVIC, M., M. PETROVIC, and M. L. LUKIC. 1982. Cellular and genetic basis of the relative resistance to the induction of experimental allergic encephalomyelitis in Albino Oxford rats. In: In vivo Immunology: Histophysiology of the Lymphoid System, Plenum Publishing Corporation, New York, p. 699.

288 15th International Leucocyte Culture Conference, Asilomar

McGill University, Montreal, Quebec, Canada

Effects of thymic epithelial cell supernatants and IL-l on the mitogenic responsiveness of thymocytes obtained from mice experiencing a graftversus-host reaction

M. L. MENDES and W. S. LAPP

Mice expenencing a graft-versus-host (GVH) reaction induced across MHC antigenic differences, demonstrate epithelial cell injury in the thymus medulla. This injury is associated with an arrest of T cell maturation. Experiments were designed to test the association between thymic epithelial cell injury and loss of T cell function. The ability of thymic epithelial cell supernatants (TES) to mature thymocytes obtained from GVH mice was studied. Also the proliferative response of GVH thymocytes to IL-l was investigated. TES was obtained from normal thymic epithelial cell cultures. GVH thymocytes were incubated with TES and either PHA or ConA for 48 hours. In order to test the proliferative effect of IL-l on thymocytes, GVH and normal thymocytes were treated in vitro with IL-l for 48 hours. Thymocyte responsiveness was measured by 3H-thymidine incorporation. GVH thymocytes demonstrated a depressed response to PHA and ConA when compared to normal thymocytes. Incubation of GVH thymocytes with TES markedly restored the ability to respond to PHA (5-7 fold) and ConA (70 fold) . TES is thus able to restore the mitogenic response of GVH thymocytes. When normal thymocytes were incubated with IL-l, cell proliferation increased 50-100 fold . However, GVH thymocytes failed to respond to IL-l. In addition, incubation of GVH thymocytes with TES failed to restore the response to IL-l . These results suggest that the acquisition of mitogen responsiveness by thymocytes is independent from the maturation of IL-l responsiveness. Thus at least two different maturation processes exist in the thymus, both of which are impaired by the GVH reaction. These results strongly support the observation that during the GVH reaction, thymic epithelial cell injury results in the arrest of T cell differentiation and thus prevents the development of competent T cells. IL-l and TES in combination with mitogen may prove useful to identify differentiative stages of T cell maturation in the thymus.

Supported by MRC and NCI of Canada.

Memorial-Sloan Kettering Cancer Center, New York, N .Y. 10021, USA

Linked production of distinct lymphokines (IL-2 and mast cell growth factor) by primary T cell microclones at limiting dilution

R. A. MILLER and o. STUTMAN

Proliferation of the mouse mast cell line 32D.c15 is dependent on a soluble factor produced by alloantigen- or mitogen-stimulated T lymphocytes (though also by the myelomonocytic leukemia WEHI-3) ; the relevant factor may be identical to Ihle's IL-3. Using proliferation of 32D.c15 cells to estimate this mast cell growth factor (MCGF) in lymphocyte-conditioned media, we have developed a limiting dilution assay for MCGF-producing T cells, and used this method to calculate the frequency of these cells in lymphocyte populations, and to measure the amount of MCGF produced by each alloantigen-stimulated helper T cell together with its immediate clonal progeny. Under our assay conditions, the limiting cell type is Thy-l + and Lyt-2-. This limiting T cell probably produces MCGF itself, rather than merely stimulating MCGF production from the . filler» cells, in that the T cell-depleted .filler» population fails to


produce MCGF even when stimulated with Con A, and in that the activity of the titrated T helper cell cannot be replaced by other lymphokines including IL-2 and IL-l. By dividing the culture supernatant from those cultures statistically likely to represent an individual microclone into aliquots which are then assayed separately for IL-2 and for MCGF, we found that all of the MCGF-producing clones also secreted IL-2. This suggests that both lymphokines may well be secreted by a single variety of T helper cell. Furthermore, the amount of MCGF produced by individual helper T cell microclones was strongly correlated with the amount of IL-2 produced by the same clones .

Supported by NIH grant AG-02479.

The University of Tennessee, Knoxville 37996, TN, USA

Enhanced responsiveness to colony-stimulating factor induced in vitro with murine interferon and lymphokine preparations

R. N. MOORE, D. W. HOROHOV, and B. T. ROUSE

Available evidence fails to suppOrt the hypothesis that myeloid colony-stimulating factors (CSF) produced in peripheral organs and tissues enhance proliferation and differentiation of myeloid precursors in the bone marrow. Rather CSF involved in myelopoiesis in the bone marrow appears instead to be produced within the bone marrow itself. Enhanced myelopoiesis in response to inflammatory and/or immunological events in sites peripheral to the bone marrow, therefore, may be dependent on signals other than CSF. Activities have been found in supernatants of polyribonucleotide-stimulated murine fibroblasts and concanavalin A-stimulated murine spleen cells that enhance the responsiveness of bone marrow-derived committed precursors to macrophage-type CSF. Murine bone marrow cells incubated overnight with the cytokine and lymphokine preparations incorporate significantly greater amounts of 3H_ thymidine than control bone marrow cells when subsequently stimulated with suboptimal concentrations of M-CSF. Enumeration of responding precursors by analysis of M-CSFinduced cloning in soft-agar cultures revealed an apparent enhancement of CSF-responsiveness induced by the cytokine and lymphokine rather than an increase in total committed precursors. The CSF-sensitizing lymphokine preparation does not contain detectable CSF activity, however, further characterization has not been performed. The cytokine has properties similar to j3 interferon (pH 2 resistant, inactivated by heating at 56 C) and can be separated from CSF by reversible binding to Affigel 202. Additionally, the CSF-sensitizing effect of the cytokine can be neutralized by addition of anti-murine j3 interferon antiserum. Finally the cytokine can be replaced with a highly purified preparation of combined a + j3 interferon (sp. activity > 5 X 108 U/mg protein). We propose that a, j3 and possibly y interferons act to enhance generation of macrophages from the bone marrow by enhancing M-CSF responsiveness of committed precursors within the marrow.

I.R.S.C., 94800 - Villejuif, France

IgG binding factor (IgGBF): characterization and binding to Ia antigens

C. NEAUPORT-SAUTES, C. RABOURDIN-COMBE, M. J. GELABERT, J. MONCUIT, and W. H. FRIDMAN

Alloantigen activated T cells (ATC) and T cell hybrids (T2D4) obtained by fusion of ATC with BW-5147 produce, upon incubation at 37°C in saline solution, a factor which binds


specifically to the Fc portion of IgG and represents the soluble form of T cell Fey receptor. This factor, named IgG Binding Factor (IgGBF) act as a non antigen specific suppressor factor of in vitro antibody production. In an attempt to characterize this factor, supernatants of ATC were passed on immunoadsorbents of sepharose 4 B coupled with Fc fragments of rabbit IgG and the material revovered in acid eluates was labeled with 1251 and analysed by SDS PAGE electrophoresis. Results show, under reducing or non reducing conditions, the presence of a single band at 60 kd MW. No band was observed in material eluted from immunoadsorbents coupled with Fab fragments of rabbit IgG. 2D gel electrophoresis reveal that IgGBF appears as a single spot (pI = 4.5) and is therefore purified to homogeneity. Since IgGBF bears Ia determinants, its relationships with Ia antigens were investigated. Immunoadsorbents coupled with glycoprotein enriched NP40 extracts of spleen cells retained both the suppressive activity and the 60 kd band characteristics of IgGBF. It could be eluted by using low pH buffer (pH 2.8) or mixture of sugars (0.1 M D glucose, 0.1 M D galactose, 0.1 Ma methyl mannoside). Both IgG and Ia antigens present in spleen cell extracts were responsible for IgGBF retention. IgGBF applied on immunoadsorbents coupled with 1. Ia antigens (purified by passage of spleen cell extracts on immunoadsorbents coupled with anti Ia antisera or anti I-A monoclonal antibodies) or 2. spleen cell extracts deprived of IgG (by absorption with staphylococcus A) could be eluted in the presence of sugars but not at low pH. Thus IgGBF purified to homogeneity binds to Fc portion of IgG and Ia antigens through two types of interactions, the first being disrupted at low pH and the second by sugars.

Department of Bacteriology Juntendo University School of Medicine, Hongo, Tokyo 113, Japan

Stimulation by lymphokine of candidacidal activity of human polymorphonuclear leukocytes

R. T. NOZAWA and T. YOKOTA

Newly developed cefem antibiotics could make it easier to treat infectious diseases caused by antibiotic-resistant bacteria, leaving an opportunistic infection by other resistant microbes such as fungi relatively more serious. We attempted to investigate the interaction of human polymorphonuclear (PMN) leukocytes from healthy volunteers with candida and the condition which encourages PMN cells to destroy infected candida cells. PMN cells were seeded in 96 flat-bottomed multiwells and infected with twofold serially diluted medically important candida. An endpoint of dilution whereby added candida cells were completely destroyed in the well was determined. C. albicans, a principal pathogen, was equally killed by PMN cells to other clinical isolates, C. tropicaiis and C. guilJiermondii. PMN cells clustered around a candida and eventually destroyed the fungi. Fungi, however, extended hyphae and outgrew PMN cells when fewer PMN leukocytes surrounded the target, indicating the collaboration of PMN cells for effective killing. In a condition where PMN cells are sparse, lymphokine (LK) that was produced by lectin-stimulated human lymphocytes increased candidacidal activity of PMN cells by 2-128 fold . The LK-treated PMN cells had a slightly stretched, irregular shape and exhibited leafy or flower-like membrane structure, while untreated cells were globular with a filament or webbing covering the surface. The stimulatory activity in LK was stable at pH 2 and undialysable. Thus far, three lymphokines affecting PMN cells are known: chemotactic factors, leukocyte inhibitory factor and eosinophil stimulation promotor. It is of interest whether our factor is the same as those. Partial purification of the factor and a search for T -T hybridoma constitutively producing the factor are in progress.


Boston University School of Medicine, Boston, MA, USA

Comparative properties of immunoregulatory alpha globulin protein and peptide in human plasma or ascites fluids

S.-K.OH

Two species of immunosuppressive substances have been identified and purified to homogeneity from normal human plasma and malignant ascites fluids. One, which is associated with large M.W. (> 200,000 daltons) complex with immunoglobulin, is an alpha-globulin factor of M.W. 50,000 dahons, exhibiting its regulatory feedback control on T-dependent lymphocyte responses in vitro and in vivo. It is trypsin sensitive protein and inhibits DNA synthesis without affecting protein synthesis on activated lymphocytes. This factor appears to be related to that of sheep erythrocyte (E)-receptor of peripheral T cells (OH, S. K., and F. L. MOOLTEN. 1981. Non-specific immunosuppressive factors in malignant ascites: Further characterization and possible relationship to erythrocyte receptors of human peripheral T cells. J. Imm. 127, 6: 2300). The other immunosuppressive factor identified from the same sources, is a small M.W. Peptide factor (M.W. 4,000 daltons), which is present in a bound form to «immunoglobulin-like» substances (cross reacts with anti-immunoglobulin) in the native state, which can be dissociated with weak acid at pH 3.0. This peptide factor is resistant to heat (100°C for 10 minutes), trypsin and carboxypeptidase B digestions, but, is sensitive to neuraminidase treatment. However, it inhibits both T -dependent and independent responses in vitro and in vivo without exerting direct cytotoxicity to lymphocytes. They both can be absorbed with solid phase anti-normal human serum and anti-lymphocyte serum. They also antagonize the effect of T-cell growth factor and inhibit natural killer cell activity on murine and human lymphoid blast cells. The former inhibits E-rosetting and can be absorbed with E, but the latter does neither. The latter can be absorbed with anti-IgG and anti-B2-microglobulin, due to its affinity to these globulins and not due to its specificity. This peptide suppressor factor appears to be similar to the lymphocyte chalone described by PRATT and HOUCK (PRATT, L. M., and J. C. HOUCK, 1980. The incredible shrinking chalone. FEBS latter 120 (2): 163).

This work was supported by NIH grant CA 15129.

Sloan-Kettering Institute for Cancer Research, Rye, NY 10580, USA

Distinct factors inducing maturation, nonspecific tumor killing, and antibody-dependent tumor killing (ADCC) in human and murine macrophages and cell lines

P. RALPH, N. WILLIAMS, I. NAKOINZ, and P. B. LITCOFSKY

Pretreatment of murine resident peritoneal or RA W264.10A line macrophages with 1 I-tg/ml LPS, 0.1 I-tg/ml tumor promoter TP A, or lymphokine (LK) greatly stimulated ADCC to myeloid and lymphoid tumors, but had no effect or inhibited nonspecific cytotoxicity to the same targets induced by LPS or TPA (Cancer Res. 41: 3546, 1981). A factor in LK (MAF) induced strong nonspecific killing of sarcoma but not of lymphoma targets without antibody (J. Immuno!., in press). LK factor stimulating ADCC was 60,000 mw and distinct from MAF

292 15th International Leucocyte Culture Conference, Asilomar

(50,000 mw) and myeloid colony-stimulating factors (with C. A. NACY and M. S. MELTZER). Fc and C receptors, latex bead phagocytosis, and Mac-l and Mac-3 antigens defined by monoclonal antibodies were induced in murine Ml myeloblast line by LPS, 10(-6)M dexamethasone, LK or WEHI-3 cytokine, but not by TPA. Ml cells did not attain cytotoxic capacity, and WEHI-3 cytokine did not induce cytotoxicity in mature macrophages. Thus, maturation and cytotoxic activities of murine macrophages appear to be regulated by separate factors, and the macrophage-related cell lines are useful model systems for studying this regulation. The same markers as for Ml were induced or increased in human U937 early monocyte line by TPA, human LK and other cytokines, but not by LPS. These agents also induced ADCC to leukemia targets in U937. Nonspecific tumoricidal activity was also induced in U937, but only by TPA. Induction of U937 to ADCC probably occurs in stages : a 40,000 mw LK induces Fc receptors and Mac-l antigen but this is not sufficient for cytotoxicity. The pattern of Mac-I, 2 and 3 antigens in other macrophage lines and induced in Ml or U937 suggests that these surface structures are not directly involved in expression of Fc receptors, phagocytosis, H202 and 02-production, 5'nucleotidase or several cytotoxic reactions. Current studies are aimed at characterization of LK factors regulating maturation and cytotoxic activities in human mononuclear phagocytes.

Rheumatology, H-811G, UCSD, La Jolla, CA 92093, USA

The magnitude of the autologous mixed lymphocyte response is a measure of T cell growth factor production

D. REDELMAN, N. ZVAIFLER, and S. NAIDES

We examined the human autologous mixed lymphocyte response (auto-MLR) by comparing the proliferative response and the amount of T cell growth factor (TCGF) stimulated in Erosetted T cells by auto non-T cells (i.e., the classic «auto-MLR»), by autologous, and by allogeneic Epstein-Barr virus (EBV) transformed B lymphoblastoid cell lines (LCL's). We found in all cases that the amounts of TCGF stimulated predicted the DNA synthetic responses. We also found that E-rosetted T cells produced a strong proliferative response when cultured alone with lectin-free TCGF, but that unseparated peripheral blood mononuclear cells (PBM) did not. We concluded from our studies that the antigens introduced by Erosetting were sufficient to induce a proliferative response in E-rosetted T cells if TCGF were provided or generated, and that the amount of TCGF generated controlled the magnitude of the proliferative response. This implies that the auto-MLR is primarily a reflection of the amount of TCGF produced. During the course of this work, we noted that B LCL's were not only effective stimulators of proliferation but also induced very high levels of TCGF production. In additional experiments, we found that B LCL's usually stimulated more TCGF production than did PHA, even with cells from donors selected for high PHA-induced TCGF generation. This was not related to the EBV immune status of the cell donors, since donors without detectable immunity to EBV produced comparable amounts of TCGF. LCL stimulation may be an effective way to produce TCGF, and the auto-LCL system should be a more defined model for examining the signals needed to induce TCGF production. For example, since T cells depleted of esterase positive cells are capable of producing substantial TCGF activity when stimulated with B LCL's, we question whether the monokine Interleukin-l is essential for this process.


Harvard Medical School, Brigham, and Women's Hospital, Boston, MA 02115, USA

Immunoregulation by polymorphonuclear neutrophils (PMN): effects of soluble products of PMN recruited by bacteria are primarily on the T cell

J. E. FITZGERALD, M. L. RODRICK, K. B. WEST, S. T. SONIS, and R. E. WILSON

The PMN has been shown to have immunoregulatory effects on both in vivo and in vitro immune responses. Furthermore, soluble products of cultured PMN (PMN-S) may have similar effects, i.e. enhancing or suppressive immune responses depending on various conditions. We report here the enhancing properties of PMN-S from PMN recruited by the bacterium Actinomyces viscosus (A V) and show its effect is primarily on the T helper cell population. PMN were recruited by intraperitoneal injection of 109 killed AV in sterile saline (0.5 ml/7-week old Balb/c mouse). Peritoneal exudate cells (> 95 % PMN) were harvested 3-4 hrs later, washed and cultured in minimum essential medium at 8 X 105 cells/ml at 37°C for 24 hrs. PMN-S was harvested following centrifugation. Study of the effect of AV PMN-S on responses of normal Balb/c splenocytes to T and B cell mitogens showed enhancing effects on T cell mitogens, when compared to control media, but no effect on response to B cell mitogen. Adherent cells were not required for the enhancing effect of A V PMN-S indicating the effect was directly on the T cell. The T cell phenotype primarily affected was found to be Lyt-I. As seen before in vivo, PMN-S enhanced both direct and indirect plaque-forming cell (PFC) responses to sheep red blood cells. However, PMN-S did not affect the polyclonal PFC response measured by Protein A plaques in response to LPS, a T cell independent response. These results indicate that PMN-S from cells recruited by AV act primarily on the T helper population to enhance responses to mitogens and antigens.

This work was supported by NIH Research Fellowship 5F32-DF 05266-02, NIH-BRSG 2-S07RR04589-19 and the Brigham Surgical Group Foundation.

Laboratory of Microbiology and Immunology, National Institute of Dental Research, National Institutes of Health, Bethesda, Maryland, 20205, USA

Monoclonal antibodies directed against human Interleukin-l

G. SCALA, C. CARTER, C. FISCHLER, R. SIRAGANIAN, and J. J. OPPENHEIM

Recent progress in production and purification of human Interleukin-l (IL-1) permitted us to attempt the production of murine monoclonal antibodies (monoAbs) against human IL-l using hybridoma techniques. Spleen cells from Balb/c mice, immunized with partially purified human IL-l, were fused with nonimmunoglobulin producing, azaguanine-resistant myeloma cells (X63-Ag8.653). After cloning (x2), and culture expansion, two hybrid cell clones, 1A5-1D6 and 2C5-1C2 that produced yG 2a and yG 2b respectively were selected on the basis of their capacity to inhibit the IL-1 enhanced proliferation of CH3/HeJ thymocytes in the costimulator assay. Cells from these clones were then injected intraperitoneally into pristane primed, 250 rad irradiated Balb/c mice to obtain a~citic antibody production. The 50 % (NH4)2S04 precipitated fraction of ascitic fluid consistently inhibited IL-1 activity by 50 % at


a protein concentration ranging from 0.1 to 16.5 tJ.g/ml depending on the activity of the batch of IL-I used in the assay. The ascitic monoAbs were further purified by DEAE sephacel chromatography. Both monoAbs eluted as a single peak at 120 m M of NaCI. In the presence of insolubilized S. aureus both monoAbs yielded better than 50 % inhibition of the bioassay at a protein concentration as low as 0.2 tJ.g/ml. These purified monoAbs were then tested for binding in Elisa assays, using human IL-I purified by AcA54 chromatography and isoelectric focusing as the antigen. Significant binding occurred at a monoAb concentration of as little as 3.0 tJ.g/ml. In order to prepare immunoaffinity columns, 2C5-IC3 DEAE-sephacel purified monoAb was coupled to CHBr-sepharose 4 B . 1000 u of partially purified IL-I were applied to this column. More than 90 % of the applied IL-l was retained by the column. Moreover, we were able to elute the about 70 % of this bound IL-l using 6M guanidine solution at pH 6.6. These results suggest that these monoAbs are directed against IL-l and may prove useful for the detection, inhibition and for immunoaffinity purification of human IL-1.

Cleveland Clinic Foundation, Department of Molecular and Cellular Biology, Cleveland, Ohio, USA

Identification of an M0 derived differentiation factor involved in CTL development

J. W. SCOTT, A. HUSAIN, M. L. HILFIKER, and J. H. FINKE

Results presented here suggest that the generation of cytotoxic T-Iymphocytes (CTL) involves a differentiation signal provided by an M0 factor in addition to the proliferative signal provided by Interleukin-2 (IL-2). We and others have reported that IL-2 is not a sufficient soluble signal for the generation of CTL. Purified IL-2, added at concentrations ranging from 0.1 to > 100 units per ml, failed to generate CTLs in a factor dependent bioassay in which nylon wool non-adherent splenocytes were sensitized to heat treated (45 °C, 60 min) allogeneic thymocytes. A factor obtained from Listeria monocytogenes (L.M.) activated M0 (plastic adherent, >95 % phagocytic and nonspecific esterase positive), did generate CTLs in this bioassay. These cells elaborate several monokines including a CTL helper factor when restimulated in vitro with heat-killed L.M. Supernates containing the M0 derived helper factor synergized with IL-2 for the generation of a much more potent cytotoxic response. The M0 factor was added to the helper bioassay in a limiting quantity so as to generate less than one lytic unit per culture. Addition of IL-2 to such cultures increased the level of cytotoxicity in a dose-dependent manner to greater than 47 lytic units per culture. In the absence of the M0 factor IL-2 did not generate a detectable cytotoxic response. This M0 factor was not active in an interferon assay and neither pH 2 treatment nor treatment with anti-Interferon y/~ affected its CTL helper activity. Biochemical evidence suggests that the M0 factor is distinct from Interleukin-l (IL-l). Although partially purified preparations of the M0 factor contain low levels of Thymocyte mitogenic protein (TMP) activity, the majority of TMP activity can be separated from the CTL helper activity by chromatofocusing. Chromatofocusing yields one major (pI 5.1-5.2) and two minor peaks of TMP activity which did not correlate with CTL helper activity. The two major peaks of CTL helper activity had pI of 4.8, and 5.0 and contained low levels of TMP activity. The pI 4.8 peak elutes from an ACA 54 gel filtration column with an apparent molecular weight of 45,000 daltons.


Rosenstiel Basic Medical Sciences Research Center, Brandeis University, Waltham, MA 02254, USA

Central regulatory circuits controlling lymphocyte and monocyte proliferation and activation

R. D. STOUT, MELISSA FISHER, and M. J. BYKOWSKY

Syngeneic spleen cell cultures generate macrophages which can effectively (> 90 % ) suppress proliferative responses of lymphocytes to mitogens and alloantigens and the generation of cytotoxic lymphocytes. Significant suppression can be detected at suppressor: target ratios as low as 1 :100 and is not mediated by prostaglandins. The suppressors inhibit the production of T cell growth and activating factors and inhibit the expression of receptors for those factors. Suppression cannot be overcome by addition of exogenous IL-1 or IL-2. However, the interleukins will prevent the generation of the suppressors. The generation of suppressor macrophages proceeds in two stages. The first stage is radiosensitive and appears to involve proliferation of nonadherent monocytes and Thy1( +) lymphocytes. The induction of the first stage requires the presence of both adherent and nonadherent spleen cells during the first 1-2 days of culture. The second stage is radioresistant, has a maximum duration of 2-4 days, and involves the maturation of nonadherent, nonsuppressive monocytes into adherent suppressors. Addition of IL-1 results in the complete inhibition of the second stage of suppressor macrophage generation. The demonstration of reciprocal interactions between central suppressors and helpers supports the hypothesis that the central suppressors and central helpers (interleukin producers and inducers) are associated as negative and positive components of central homeostatic regulatory circuits controlling lymphocyte and monocyte proliferation and activation.

Dept. of Microbiology and Immunology, Univ. of Ill., Medical Center, Chicago, Illinois 60612, USA

Self-induction of a polyclonal B cell activator (PBA) in high density lymphoid cell cultures

M. TEODORESCU, J. L. CHANG, S. DRAY, and D. GANEA

Evidence presented here suggests the following mechanism of T -B cell collaboration in antibody formation. The role of the antigen is to bring in close contact B and T cells which otherwise have the tendency of homing in different areas of the lymphoid organs. Due to their close contact, B cells stimulate T cells polyclonally; the stimulated T cells produce a PBA, which is an o2macroglobulin (o2M) bound protease. This PBA activates the B cells which will produce the antibody against the involved antigen. To demonstrate this model, rabbit lymphoid cells (RLC) were cultured in a serum free medium, 106 cells/ml, and the medium was changed every day for three days. The RLC were incubated in parallel in polystyrene flasks in two positions; a) standing to obtain a low cell density and b) tilted to obtain a high cell density. Thus, the difference between the two types of cultures was only the position of the vessel in


the incubator. From these cultures we obtained low and high density supernates (lDS and HDS) respectively. To demonstrate the presence of a PBA in supemates we used three basic methods: 1. The Ig-turnover assay, 2. The induction of blast transformation measured by 14C_ uridine or 3H-TdR uptake, and 3. Induction of antibody formation in nude mouse spleen cell cultures in the absence of antigen. We found that 1. The PBA was present in HDS but not in lDS; 2. The PBA was present when Band T cells were present together; 3. The PBA titer and the aM concentration was similar in all three consecutive HDS; 4. The PBA activity was associated with aM only; 5. The PBA activity of the HDS or of the aM from HDS was blocked by aprotinin and other low molecular weight protease. inhibitors; aprotinin had no effect on RLC stimulation by LPS or dextran sulfate; 6. In cultures of allogenic B and T cells the a 2M-PBA had the z allotype of the T cell donor; 7. HDS but not LDS induced antibody forming cells against SRBC; 8. Pure thoracic duct lymphocytes produced and responded to the PBA from HDS. These observations are consistent with the hypothesis that antigens can trigger an in vivo autologous MLR; it need not stimulate B or T cells, directly.

Supported by NIH Grants AM28469 and AI07043.

Scripps Clinic and Research Foundation, La Jolla, CA, USA

Synergy between a T cell-replacing factor and IL-2 B cell growth factor

MARILYN L. THOMAN and W. O . WEIGLE

A T cell-replacing factor (Fc-TRF) is produced by Lyt 1 + 2 - murine T lymphocytes upon stimulation with Fc fragments of immunoglobulin or Con A. This factor was originally characterized as replacing T cell function in the induction of a polyclonal antibody response triggered by Fc fragments. Further investigations of the mecharnism of action demonstrate that Fc-TRF has several activities and may act in synergy with both Interleukin-2 (IL-2) and a recently described B cell growth factor (BCGF). Fc-TRF cannot trigger a polyclonal response alone, but rather acts secondary to a stimulation agent such as Fc fragments or a short LPS pulse, driving the differentiation of activated B lymphocytes to antibody-secreting cells. No poly clonal antibody is produced if Fc-TRF is added prior to Fc fragments. However, the reverse order of addition, Fc fragments followed by Fc-TRF yields a significant response, and Fc-TRF addition may be delayed up to 48 hours after the Fc fragments and still be effective. Fc-TRF does not replace helper T cells in the generation of anti-SRBC PFC; however, it synergizes with IL-2 to reconstitute this response. Under the conditions employed, Fc-TRF induces no PFC, while IL-2 triggers very few (120 PFC/ I06 ). Addition of both factors together markedly enhances the response, increasing by 6-fold the number of PFC generated (700 PFCI lQ6). Further, Fc-TRF synergizes with a B cell growth factor, augmenting the proliferation of the B leukemic cell line, BCl l . Both BCGF and Fc-TRF alone stimulate BCL I growth. However, together the enhancement of BCll proliferation is significantly greater than additive. Fc-TRF can stimulate 3H-thymidine incorporation by splenic B lymphocytes, but BCGF cannot. These studies indicate that Fc-TRF delivers one of several signals necessary for driving the multi-step process of activation and differentiation of B lymphocytes to antibodysecreting cells.


UCLA School of Medicine, Los Angeles, CA 90024, USA

Non Interleukin-2 T cell growth factor

CHRISTEL UITIENBOGAART and J. FAHEY

In previous work (1) we have shown that the human malignant T leukemia cell line MOLT-4f produces a growth factor to which it also can respond. This finding is in agreement with the premise, suggested by TODARO for sarcoma virus transformed cells (2) that some malignant cells are independent, since they make a growth factor for which they also have receptors. The growth factor produced by MOLT-4f, which we designated Leukemia Derived Growth Factor of MOLT-4f (LDGF-M4) differs from Interleukin-2 (IL-2) in biological and physicochemical properties. A similar growth factor activity has been found in the supernatant of the T leukemia cell lines CCRF-HSB-2 and CCRF-CEM. In order to determine if «fresh» leukemia cells produce a growth factor for MOLT-4f cells, we studied the effects of supernatants collected from freshly obtained leukemia cells on the growth of MOLT -4f cells. T lymphoma and nonTnonB leukemia cells were separated on Ficoll Hypaque, washed and resuspended in serum free medium (Iscove's modified Dulbecco's medium supplemented with bovine serum albumin and transferrin). The cells were cultured in this medium, the supernatants were harvested and filtered after 48 hrs and tested for their growth-promoting effects on MOLT-4f cells. Supernatans of 3 T lymphoma and 2 nonTnonB acute lymphocyticleukemias contained growth factor activity, which was not IL-2. This indicates that T lymphoma and nonTnonB leukemia cells make a growth factor similar to LDGF-M4.

1. UITIENBOGAART, C. H., and J. L. FAHEY. 1982. Soluble growth factors (non Interleukin-2) (IL-2) produced by malignant lymphoid cells. Proc. AACR 23: 1032.

2. TODARO, G. J., and J. E. DE LARCO. 1978. Growth factor produced by sarcoma virustransformed cells. Cancer Res. 38: 4147.

Supported by grant CA 12800 of the National Institute of Health and by the Concern Foundation.

Medical Biology Institute, La Jolla, CA 92037, USA

The construction and characterization of murine T cell hybridoma lines which secrete constitutively B-cell-specific immunoregulatory factors

R. G. WEINER, KAREN L. EVANS, A. D. SFERRUZZA, E. FERNANDEZ-CRUZ, D. H. KATZ, and A. ALTMAN

The studies described herein were designed to: a) establish homogeneous cellular sources of B-cell-specific immunoregulatory factor(s) (BCSF) devoid of T cell growth factor (TCGF) activity, and b) devise strategies which will allow us to distinguish between growth and differentiation-promoting activities of BCSF. Murine T cell hybridoma lines were constructed by fusing alloactivated T cell blasts with the HAT-sensitive BW5147 T lymphoma line. Supernatants of unstimulated or mitogen-stimulated cloned hybridomas were initially screened for stimulation of primary in vitro antibody responses to TNP-SRBC by T celldepleted spleen cells. Several clones were found to secrete constitutively BCSF by this criterion. Several, but not all, of these clones displayed I_Ad determinants detected by a monoclonal antibody. No TCGF activity could be detected in any of the biologically-active supernatants tested to date. In order to ascertain the secretion of B cell growth factor(BCGF)like molecules by these T hybridoma lines, we tested the effects of their consitutive superna-


tants on the growth in culture of an ascites variant of the anti-DNP IgA-secreting plasmacytoma, MOPC-315, which nonnally fails to grow in vitro. Some hybridoma supernatants as well as conventional allogeneic effect factor (AEF)-containing culture supernatants were found to markedly promote the growth of MOPC-315 cells in vitro, and these effects could be detected with as little as 100 tumor cells per well and as early as 24 hours after stimulation. We are now developing bioassays for differentiation-promoting activities by measuring changes in the production of anti-DNP myeloma proteins by cultured MOPC-315 cells. Such hybridoma lines and bioassays should provide us with important tools to analyze the precise role of Tcell-derived lymphokines in the regulation of B lymphocyte activation, differentiation and growth.

IInstitut Curie (Paris), 2IRSC (Villejuif), 3Hopital Avicenne (Bobigny), France

Studies on purification of human lymphokines and gamma interferon

J. WIETZERBINI, E. P. KOLB2, A. SENIK2, L. DER STEPANI1, J. SANC£AU\ G. ANDREU3,

R. FALCOFF\ E. FALCOFF1

We have devised a method for producing large amounts of human y interferon in lymphocyte cultures stimulated by PHA. Titers were usually in the range of 10,000 to 30,000 u/ml. Crude y interferon can be absorbed on silicic acid, from which the antiviral activity is eluted by a buffer containing ethylene glycol and a high salt concentration. This treatment allows quantitative recovery of y interferon with a specific activity of 5 X 105 to 1 X 106 u/mg of proteins. Chromatography on several sorbents (Blue Sepharose, Remazol blue agarose, CM Sephadex, Blue trisacryl, Decyl agarose, poly I-agarose, Sephacryl S 200) of silicic acidpurified y interferon as well as chromatofocusing analysis enabled us to ascertain some of the physicochemical properties of y interferon, including molecular heterogeneity, isoelectric points, molecular weight and apparent hydrophobicity. By combining some of these methods, y interferon was purified to a specific activity of 1-5 X 107 u/mg. We also tested y interferon preparations for lymphokine activities (such as B cell helper factors and TCGF) during different fractionation steps. Such activities were present in y interferon preparations eluted from silicic acid, and they were chromatographed together with the antiviral activity in several sorbents. Our results suggest that y interferon and lymphokine activities shared some physicochemical properties such as affinity for the Blue chromophore Cibacron F3GA and for polynucleotides. Preparations of y interferon purified to a specific activity of 107 u/mg still contained Iymphokine activities. However, taking advantage of their different isoelectric points, we achieved dissociation of lymphokines and antiviral activity by chromatofocusing. Such dissociation is a pre-requisite to the study of the biological properties of these molecular entities.

Dept. of Pathology, The Johns Hopkins University School of Medicine, Baltimore, Md. 21205, USA

Alphat-antitrypsin modulates biological activity of interleukin-l

W. F. WILLOUGHBY, J. B. WILLOUGHBY, and P. L. SIMON

Interleukin-1 (IL-1) is a macrophage-derived protein which 1. enhances T-cell responses to mitogens and antigens by causing T helper cells to secrete IL-2; 2. stimulates synovial cells to


produce collagenase; 3. stimulates liver cells to produce serum amyloid A; and 4. stimulates fibroblast proliferation. We have further shown that rabbit alveolar macrophages stimulated in vitro by LPS produce two distinct species of IL-I possessing pI values of 4.6 and 7,4, and molecular weights of 14,400 and 11,600, respectively, and which are identical to the endogenous pyrogens produced in vitro by rabbit peritoneal macrophages. We now show that when rabbits inhale LPS they develop fever and pulmonary interstitial inflammation. Alveolar macrophages obtained by bronchopulmonary lavage during the febrile response are observed to be releasing large quantities of IL-I into the fluid phase, as measured by both thymocyte and fibroblast proliferation assays. Both of these biological activities are inhibited by commercial human alphal-antitrypsin (A 1-AT), and also by partially-purified rabbit AI-AT, in a dosedependent fashion. Such inhibition requires «acute-phase. concentrations of AI-AT (2-4 mg/ mI) and does not result from direct toxic effects by AI-AT on cells. We propose that alveolar macrophage IL-I plays a central role in mediating pulmonary inflammation, and that, under normal circumstances, such inflammation is «self-limited. through increases in «acute phase» serum proteins. We further propose that AI-AT, in particular, acts to down-regulate the initiation of inflammatory reactions through its action on IL-I, in addition to limiting tissue damage by extracellular proteases.

Arbeitsgruppe Biochemie, Max-Planck-Institut fUr Physiologische und Klinische Forschung, Benekestrafie 2, D-6350 Bad Nauheim, FRG

Leukocytic polypeptide mediators and hormones controlling tissue morphogenesis

J. H . WISSLER

The protein and cell constituents of blood as sources of humoral and cellular effector molecules of inflammation and wound healing playa prominent role in the chemical activation and regulation of tissue regeneration and morphogenesis processes. Effectors represent activating and inhibiting chemical signals for operation of basic chemical mechanisms for cellular reactions and communication in tissue morphogenesis. Six basic categories of chemical mechanisms controlling these reactions are known to organize molecular structures and cells in tissue morphogenesis. These are chemopoiesis, chemorecruitment, chemokinesis, chemotaxis, chemotropism and chemostasis of cells. For each of basic categories of mechanisms controlled by chemicals, distinct cytokine effector polypeptides of high biological (cell and reaction) specificity can now be shown to exist. Biotechnical methods have been established (I) to isolate such chemical trace signal polypeptides for the first time in a high degree of molecular homogeneity in physical (mg) amounts for functional, clinical and structural studies. Sources for isolation are inflammed tissue sites (infarcted heart muscle), serum and (-1000 I) supernatant solutions of large scale, serum-free cultures of (-50 kg £!, -1014) leukocytes (granulocytes, monocytes, lymphocytes) which must be isolated from a total of -10,000 I porcine blood. Autocrine and paracrine mechanisms of information transmission by leukocytic mediators (leukokines, monokines, Iymphokines) are exemplified by optimum morphogenesis of blood vessel patterns with active hemodynamics by angiotropin polypeptides (I) as blood vessel (endothelial) cell growth factors above thresholds of 2 fmol in ocular (cornea), embryonic (chorio-allantois) membrane and skeletal muscle tissues. Leukocytes also may control distinct steps of regeneration and morphogenesis processes and the formation of a secretory leukocytic tissue (I) by endocrine functions (e.g. chemorecruitment), as first shown by the existence of true wound hormones (I). Their demonstration proves leukocytes as true unicellular, mobile endocrine gland cells. Further effectors isolated are operating leukopoiesis, -leftward shift, kinesis, -taxis and thermoregulation (fever).

1. WISSLER, J. H. 1982. In Jaenicke, L. (ed.): Biochemistry of Morphogenesis, Springer.


Jefferson Medical College, Philadelphia, PA 19107, USA

Helper factors distinct from IL-2 in the autologous mixed lymphocyte reaction (AMLR)

J. Wows and J. BRUCE SMITH

The AMLR measures a proliferative response of T cells to I region determinants on autologous or syngeneic non-T cells. Cytotoxic activity is not normally generated. However, the addition of trinitrophenyl-modified, mitomycin c treated syngeneic thymocytes (TNP-T m) to a murine AMLR results in a TNP specific, H-2 restricted, cytotoxic response. In the absence of an la bearing stimulator cell, cytotoxicity does not develop. The cytotoxic potential can be restored if supernatants from AMLR cultures are added to cultures of T cells + TNPT m prior to incubation. The active factor in these supernatants appears to be distinct from IL-2 and IL-l. These AMLR supernatants are unable to maintain an IL-2 dependent cell line and fail to act as a costimulator in assays detecting IL-1 and IL-2. Unlike IL-2, AMLR supernatants cannot activate cytotoxic precursors in the thymocyte population, mature T -cells need to be present. The mediator is produced by an Lyt1 + 23 - T-cell and is H-2 unrestricted. If the thymocyte population is passed over a nylon wool column prior to hapten modification, the AMLR supernatants do not manifest their activity. The evidence suggests we are dealing with a soluble mediator which may play a role in the initiation of the cytotoxic response. In the presence of this factor and hapten modified thymocytes, thymic adherent cells could be induced to form IL-1, which could activate helper/inducer T cells to make IL-2. The IL-2 would bind to receptors on antigen-sensitized cytotoxic T cell precursors and lead to the differentiation of antigen-specific cytotoxic effector cells.

Department of Immunology, University of Occupational and Environmental Health, School of Medicine, Kitakyushu 807, Japan

Suppressive effect of phorbol myristic acetate (PMA) on the induction of cytotoxic T cells in vitro

U. YAMASHITA

Immune response can be modified by several biological active substances. Recently, some attention has been focussed on PMA as a potent modifier of immune responses, because it can stimulate lymphocytes from several species to proliferate and can replace macrophage function in the T cell activation such as lectin-induced proliferation and interleukin-2 production. In order to further analyze the activity of PMA on the immune response, the author studied the effect of several phorbol esters on the proliferative response and the induction of cytotoxic T cells in vitro using mouse spleen cells. PMA stimulated spleen cells to proliferate at concentrations of 1-1000 ng/ml. Other phorbol esters such as phorbol, phorbol monoacetate and phorbol monomyristate had no stimulating activity. Responder cells to PMA stimulation were T cells, but T cells alone could not respond to PMA and accessory cells were required. The accessory cells were la-positive macrophages. The stimulation activity of PMA was replaced by soluble factors elaborated by spleen cells treated with PMA. Furthermore, T cell stimula-


tion with soluble factors also required the presence of macrophages. By a gel filtration these factors were fractionated into three major peaks with a molecular weight of 70K, lOOK and 200K daltons. PMA at the same concentrations suppressed the development of hapten-reactive cytotoxic T cells. The brief treatment of spleen cells with PMA induced suppressor T cells which suppressed the development of cytotoxic T cells. This suppressive effect was also replaced by soluble factors elaborated by spleen cells treated with PMA. By a gel filtration the suppressive factors were fractionated into the same fractions of the stimulating activity of proliferative response. Thus, PMA has two activities on the immune response: One is a stimulation of T cell proliferative response and the other is a suppression of the development of cytotoxic T cells and these activities seem to be mediated by similar factors.

Medical College of Virginia, Richmond, VA 23298, USA

Macrophage-T cell interactions in the induction of immune interferon (IFNy) in asymptomatic carriers of hepatitis B virus

S. YOUSEFI and M. R. ESCOBAR

The persistence of hepatitis B virus (HBV) in some individuals after acute infection could be due to a deficiency in one or more host factors of cellular immunity. Our earlier studies showed that IFN-y upon stimulation by hepatitis B surface antigen (HBsAg) was absent in asymptomatic carriers of HBV. However, INF-y induction by polyclonal activators (e.g., PHA) was normal in these patients. These findings led us to investigate the ability of human macrophages derived from normal donors or from asymptomatic carriers of HBV to enhance in vitro T lymphocyte IFN-y production as a measure of the functional integrity of the patients' macrophages. Macrophage competence to interact with T lymphocytes and thereby enhance the level of IFN-y was tested by a micromethod. Autochthonous macrophagelymphocyte cultures were made from normal subjects or from asymptomatic carriers of HBV; or allogeneic cultures were prepared in which macrophages from normal subjects or from individuals who had recovered from HBV infection were combined with the patients' T lymphocytes, and vice versa. For determining the level of IFN, several cell lines were compared for their sensitivity to IFN-y by a microtiter dye-binding assay. Preliminary results indicate that selective deficiency of asymptomatic carriers of HBV to produce IFN-y may be related to the functional integrity 0 their macrophages. The correct interpretation of our findings is contingent on further dissection of the system to identify possible functional differences of both macrophage and lymphocyte subpopulations.

workshop 11 a and b lymphokines

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