SECOND INTERNATIONAL WORKSHOP ON CYTOKINES / 109

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ENHANCEMENT OF IL-l AND TNF PRODUCTION BY BRADYKININ. Edward 5. Kimball. Dept. of Biological Research, Janssen Research Foundation, Spring House, PA 19477, USA.

i'llNOR NECROSIS FACTOR a mRNA EXPRESSION AND RELEASE BY HUMAN EPIDERNAL CELLS. A.Kijck', A.Urbanski' , T.Schwarz-, T.A.Luqer'; 'Dept.Derm.11, Univ.Vienna and LBI-DVS, Lab.Cell- biol. Vienna; "Dept.Derm.Hosp.Lainz, Vienna, Austria

Studies conducted in our laboratory suggest that inflammatory processes can be exacerbated by interactions between vasoactive peptides, cells which produce cytokines, and their cytokine products. Previously, we demonstrated that substance P (SP) and bradykinin (BK) each acted synergistically with interleukin-1 (IL-I) using BALB/c3T3 fibroblast multiplication as a node1 for synovial hypertrophy (J. lmmunol., 141: 3564. 1988). The combination of either SP or BK with IL-1 womoted an increase in cell number that was not observed when any of the agents were used alone. We also examined that production of IL-1 by the P38BDl nwuse macrophage cell line in response to SP, and showed that SP caused these cells to produce IL-1 activity (J. In~~~nol., 141: 4203, 1988). More recently we examined the effect of BK on IL-1 and TNF production. BK was tested in vitrq on thioglycollate elicited mouse peritoneal macrophages. We observed approximately a Z-fold increase in the levels of IL-l and TNF activity secreted into culture supernatants when I-100 nH BK was added to cultures containing LPS. Neutralization assays provided imnunochemical evidence for the identity of the cytokines ds IL-1 or TNF, respectively. Lower levels of IL-l activity were observed in supernatants from cells not cultured with LPS. These results further support the hypothesis that cytokines and vasoactive peptides can interact to promote and sustain inflammatory responses, and may be elements of a mutual positive-feedback system.

Tumor necrosis factor-a (TNFa) in addition of being cytoto- xic for certain tumors, like IL 1 and IL 6 has been shown to be a multifunctional cytokine which is involved in the regu- lation of immunity and inflammation. Since epidermal cells (EC) are known to produce many cytokines including IL 1 and IL 6 in the present study it was investigated whether human long term cultured EC and epidermoid carcinoma cell lines (A431, KB) produce TNFa. TNFa was measured either by a TNFa ELISA or in a cytotoxic assay using L929 cells. EC and KB cells constitutively only produced low levels of TNFa. How- evei-, in the presence LPS (100 pg/ml), IL 6 (10 U/ml) or upon UV irradiation (100 J/cm?) a significant increase of EC TNFa production was observed. In contrast, IL la or IL 1R failed to indcue EC TNFa production. Maximum TNFa production was observed 12 hr after treating 1x10* KB cells with 10 U/ml IL 6. Upon HPLC gel filtration and Western blot analysis EC derived TNFa exhibited a m.w. of approximately 17 kD. More- over. Northern blot analysis was performed using a TNFa cDNA probe. TNFa mRNA only was detected in EC following stimula- tion with LPS or IL 6. These findings indicate that EC upon stimulation are capable to produce TNFa which in concert with other epidermal cell derived cytokines may mediate host defense against microbial agents and tumors.

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DIFFERENTIAL REGULATION OF IL-1 AND TNF PRODUCTION BY LEVANISOLE. Edward S. Kimball, N. Christina Clark, Craiq R. Schneider, N. Carolyn Fisher and Francis J. Persico. Janssen Research Foundation, Spring House, PA 19477, USA.

CYTOKINE REGULATION OF ICAM- EXPRESSION ON HUMAN GLIOBLASTOMA CELLS. M.C. Kuppner, M.F. Hamou, E. van Meir, N. de Tribolet. Neurosurgical Service, Univ. Hospital, CH-1011 Lausanne, Switzerland.

Recent studies to elucidate the mechanisms by which levamisole may modulate itmnune responses have focused on cytokine production by bacterial lipopolysaccharide (LPS)-activated macrophages. u vitro treatment of the P38801 muse macrophage cell line with LPS in the presence or absence of levamisole for 18-24 hours revealed that levels of interleukin-I (IL-I) secreted during a subsequent 24 hour period were enhanced when the LPS-stimulated cells were co-cultured with levamisole (1 j&n). Levamisole, however, did not directly stimulate cells to secrete IL-1 in the absence of LPS. These results were confirmed and extended b

m using thioglycollate-elicited mouse peritoneal macrophages (PM). 1_? e administration of a single dose of levamisole (3 mglkg, p.o.) also resulted in enhanced IL-1 secretion by elicited PM's when the cells were stimulated in vitro with LPS. In contrast. TNF activitv was diminished in supernatants from cultures of LPS-stimulated cells taken from levamisole-tl,eated mice compared to control mice. TNF inhibitory activity was not observed in the culture supernatants. Elicited PM's from mice to whom levamisole was administered, but which were not stimulated b w with LPS, secreted low, but measurable levels of IL-1 activity, and no measurable TNF activity. These results suggest that one mechanism by which levamisole acts to normalize ilnmune responses may be to provide complex second signals which enhance the ability of activated macrophages to secrete IL-I, but which have differential effects on other cytokines.

Intercellular adhesion molecule 1 (ICAM-1) is a ligand for lymphocyte function associated antigen 1 (LFA-1). Imnunohisto- chemical staining of frozen tissue sections using the ICAM-I antibody RR1/1 demonstrated significant levels of ICAM-I expression on human glioblastoma cells and on intratumoral vascular endothelial cells. ICAM-I was either weakly expres- sed or absent from low grade glibmas and absent from normal and fetal brain. Surface antigen expression of ICAM- and ICAM-I specific mRNA could be significantly increased by incubating glioblastoma cells with TNFct, IFNXd'; and ILIP whereas ILZ, IL4, IL6 and TGFBZ had no effect. TNFaand ILlg at l-10 U/ml gave similar levels of enhancement of ICAM-I expression whereas higher concentrations of IFNX(500 U/ml) were required to give a similar effect. ICAM- expression steadily increased with cytokine treatment for up to 48h. Supernatants derived from LAK cell cultures also had a potent enhancing effect on ICAM- expression. Increased ICAM-I expression on glial cc!ls and endothelial cells ms: be related to iho increase in cerebral edema observed after LAK cell therapv.

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TNF RESISTANCE CORRELATES TO FREE RADICAL RESISTANCE. K. Sean Kimbro and M.W. Taylor. Indiana University, Bloomington, IN 47401 ME-180 (human cervical carcinoma cell line) cells resistant to tumor necrosis factor-alpha (TNF-alpha) were Isolated by gradually increasing the TNF-alpha levels over time. The cell lines were cloned and partially characterized. The resistant clones display resistance to TNF and free radicals generated by xanthine oxidase. In the presence of 1000 units TNF-alpha/ml for 72 hours, resistant clones were greater than 80% more resistant than the parental line as determined by dye exclusion assays. In the presence of 0.5 microunits of xanthine oxidase the resistant clones are approximately 60% more resistant than the parental line. It has been shown that there was approximately a two-fold increase in Cu/Zn superoxide dismutase and catalase mRNA after 24 hour exposure to 1000 units/ml TNF. It should be noted that there is approximately a two-fold increase in Cu/Zn SOD in trisomy-21 individuals and a high incidence of leukemia. These data suggest a correlation between TNF cytotoxicity and free radical production. Further studies with ME-180 transfected with Cu/Zn SOD cDNA and TNF- resistant ME-180 are underway.

EFFECT OF AFLATOXIN Bl (AFBl) ON IL-1 PRODUCTION BY BOVINE MONOCYTES AND MACROPHAGES. R. Kurtz and

. Czuorvnski Sch. Vet. Med, Univ. Wise, Madison, WI 53706. Contamination of feed and foodstuff with secondary metabolites

(aflatoxins) produced by fungi in the genusAsp@& causes morbidity and mortality in man and animals. Chronic low level exposure have been reported to induce increased susceptibility to infectious agents. Attempts to correlate diminished resistance to infection with impaired immune function have met with limited success. In this study we examined the effects of preincubation with AFBI on the ability of bovine monocytes and macrophages to produce IL-I when stimulated with bacterial lipopolysaccharide (LPS). Culture supematants were assayed for IL-1 activity in the C3H/HeJ thymocyte costimulation assay. Following an 18h preincubation with 10 @ml AFBl, blood monocytes and alveolar macrophages had a markedly diminished capacity to release IL-1 activity in response to LPS. Preincubation with 10 @ml for only lh or for 18h with lower concentrations of AFBl did not affect IL-1 production. AFBI had similar effects on monocyte production of IL-1 activity when other inducing agents, such as Listei-ia monocytogenes, were used. Attempts to correlate the diminished IL-1 activity following aflatoxin pretreatment, with the level of transcription of IL-1 mRNA, are underway. These data support the hypothesis that the immunosuppressive effects of aflatoxins may in part be attributable to diminished monokine production.