immunoreactivity for il-1 beta and tnf alpha in human lymphoid

AmericanJournal ofPathology, Vol. 135, No. 5, November 1989

Copyright X American Association ofPathologists

Immunoreactivity for IL-1 Beta and TNF Alpha inHuman Lymphoid and Nonlymphoid Tissues

Luigi P. Ruco,* Antonella Stoppacciaro,*Donatella Pomponi,* Diana Boraschi, AngelaSantoni,t Aldo Tagliabue, Stefania Uccini,*and Carlo D. Baroni*From II Cattedra diAnatomia Patologica, DipartimentodiBiopatologia Umana,* and ICattedra diImmunologia,Dipartimento di Medicina Sperimentale,j Universita'"La Sapienza, "Roma, and Laboratorio diImmunofarmacologia, Centro Ricerche Sclavo,Siena,j Italy

Monoclonal antibodies (MAbs) against two non-

cross-reacting antigens ofhuman IL- I beta (Vhp20and BRhC3) and human TNF alpha (B154.2 andB154. 7) were applied to identify cytokine-contain-ing cells in tissue sections and in cell suspensions.IL- I beta- or TNF alpha-positive cells were notpres-ent in immunostained cytocentrifuge smears pre-

paredfromfreshly isolatedperipheral blood leuko-cytes, spleen, and lymph node cells. After 18 hours

ofculture with bacterial endotoxin (LPS), 80% to90% of blood monocytes, 30% of spleen macro-

phages, and 2% to 28% of lymph node macro-

phages were strongly positivefor IL- I beta with ei-ther of the MAbs. Furthermore, 25% to 35% ofblood monocytes and 6% to 60% of lymph nodemacrophages were stainedfor TNFalpha. Cellspos-itivefor IL- I beta or TNF alpha were extremely rare

in sections of normal thymus, spleen, and lymphnodes. ImmunoreactivityforIL- I beta or TNFalphawasfrequently observed in sections ofgranuloma-tous lymphadenitis (N = 11). IL- I beta or TNF al-pha staining was confined to the epithelioid macro-phages forming the granuloma, and the intensityofTNF alpha reactivity was generally stronger. Thehighfrequency ofcytokine-containing cells in thispathologic condition was confirmed in a cell sus-

pension study showing that 20% of epithelioidmacrophages were weakly positive for IL-I betaand 809% were strongly positivefor TNF alpha. Thepresence of cytokine-containing cells was investi-gated in cryostat sections of several nonlymphoidorgans with normal histologic appearance. IL-i

beta reactivity was not observed in any of the tis-sues. TNF alpha reactivity was frequently demon-

strated in isolated macrophages embedded in theinterstitial connective tissue. (Am J Pathol 1989,135:889-897)

Tumor necrosis factor alpha (TNF alpha) and interleukin 1(IL-1) are soluble factors that play a pivotal role in acuteand chronic inflammation. TNF alpha is a 17 kd proteinwith an aminoacid sequence identical to that of cachectin.TNF alpha/cachectin is mainly released by monocytesand macrophages, and is a common mediator of toxicshock, cachexia, and tumor necrosis (for review see refer-ence 1). IL-1 was first described as a lymphocyte-activat-ing factor for its ability to costimulate mitogen-activatedmouse thymocyte proliferation.2 It was then discoveredthat IL-1 has a number of other biological activities andthat there are at least two major types of IL-1 (alpha andbeta),3 which bind to the same receptor.45 In addition tothe effect on T lymphocytes, IL-1 is able to stimulate Bcell functions, fibroblast proliferation, muscle proteolysis,prostaglandin E2 and collagenase production, and he-patic acute-phase protein synthesis, and to induce fever(for review see references 6 and 7).

TNF alpha/IL-1 beta synthesis and release have beenextensively investigated in in vitro systems by the use ofbiological assays, radioimmuno assays, immunoblot, andNorthern blot analysis. The optimal conditions for TNF al-pha release include monocyte and macrophage primingwith T lymphocyte products like interferon-gamma or GM-CSF and subsequent triggering with bacterial endo-toxin.8-10 These conditions closely resemble those pre-viously described to induce tumoricidal macrophages."Monocyte/macrophages are also major producers of IL-1beta in response to bacterial endotoxin, interferon-gamma, and GM-CSF.67'10 Thus, TNF alpha and IL-1 betaseem to be crucial molecules in the regulation of lympho-cyte-macrophage interactions.

In spite of the extensive information concerning thebiochemistry and the cell biology of TNF alpha and IL-1beta, very few studies have addressed the demonstration

Supported by a grant from Istituto Pasteur-Fondazione Cenci-Bolognetti,Roma, Italy.

Accepted for publication July 7, 1989.Address reprnt requests to Luigi P. Ruco, MD, II Cattedra di Anatomia

Patologica, Dipartimento di Biopatologia Umana, Universita' "La Sa-pienza", Viale Regina Elena 324, 00161 Roma, Italy.

889

890 Ruco et alAJPNovember 1989, Vol. 135, No. 5

of cytokine-containing cells in normal and pathologic tis-sues.12-15 We approached this problem by the use of anti-TNF and anti-IL-1 monoclonal antibodies and enzyme im-munocytochemistry. By this technique, the presence ofTNF alpha- and IL-1 beta-containing cells was investi-gated on sections and on cell suspensions obtained froma variety of lymphoid and nonlymphoid tissues. Our find-ings indicated that enzyme immunocytochemistry is a reli-able and promising technique for investigating the poten-tial role of cytokines in the development of the tissue le-sions.

Materials and Methods

Tissues and Cell Suspensions

Fragments of lymph nodes, spleen, thyroid, lung, skin,and breast were removed at surgery for diagnostic pur-pose. Fragments of fetal thymus, adenohypophysis,adrenal, esophagus, kidney, heart, and brain were ob-tained at autopsy. Surgical and autopsy specimens wereembedded in OCT compound (Ames Division, Miles Lab-oratories, Elkhart, IN), snap-frozen in liquid nitrogen, andstored at -80 C until sectioning.

Lymph node and spleen cell suspensions were ob-tained by gently scraping the tissues with a scalpel. Pe-ripheral blood leukocytes (PBL) were obtained from nor-mal volunteers and were isolated by centrifugation on dis-continuous gradient. PBL, spleen cells, and lymph nodecells were suspended in RPMI-1640 medium containing1% human AB serum, 100 U/ml penicillin, and 100,ug/mlstreptomycin. A fraction of the cells was used to preparecytocentrifuge smears; another fraction was cultured in50 ml polypropylene test tubes at a concentration of 2X 1 06/ml with 20 sg/ml LPS from Escherichia coli 055.B5(Sigma Chemical Co, St. Louis, MO) for 18 hours in anatmosphere containing 5% CO2 at 37 C. At the end ofincubation, LPS-stimulated cells were cytocentrifuged.Cytosmears were individually wrapped in silver paper andwere stored at -80 C until immunostaining.

Immunostaining Procedures

Cryostat sections and cytocentrifuge smears were fixedin acetone for 10 minutes at room temperature and wereimmunostained with monoclonal antibodies to IL-1 beta,TNF alpha, and monocyte and macrophage antigens. IL-1 beta was demonstrated with Vhp2O, a monoclonal anti-body to the synthetic IL-1 beta peptide 163-171,16 andwith BRhC3, a mouse monoclonal antibody obtained byimmunization with recombinant IL-1 beta that recognizesan epitope within the fragment in position 133 to 147 inthe IL-1 beta (provided by M. Bigio and R. Rossi, Sclavo,

Siena).17 TNF alpha was recognized by B154.2 andB154.7 (noncross-reacting mouse monoclonal antibodiesdirected against two distinct determinants of TNF alphathat do not react with lymphotoxin), whose specificity wasdescribed previously.18 B154.2 and B154.7 were donatedby B. Perussia, Wistar Institute, Philadelphia, PA. Mono-clonal antibodies Vhp2O, BRhC3, B154.2, and B154.7were obtained as Durified ascites fluids. Monocyte andmacrophages were demonstrated by Dako-mac (Dako-patts, Geostrup, Denmark).

Cryostat sections and cytosmears were preincubatedwith normal horse or goat serum to prevent nonspecificbinding, and then incubated with an optimal dilution of theprimary antibody (Vhp2O, BRhC3, B154.2, and Bi 54.7, 1:100 v/v; Dako-mac, 1:10 v/v) for 30 minutes. The slideswere sequentially incubated with biotin-conjugated horseanti-mouse immunoglobulin antibodies followed by avid-in-biotin-peroxidase complex (PK 4002; Vector Labora-tories, Burlingame, CA) or by streptavidin peroxidasecomplex (Amersham, Little Chelfont, UK). In some experi-ments a peroxidase-labeled mouse immunoglobulin(Cambridge Research Laboratory, Cambridge, MA) (PAPmethod). Each incubation step lasted 30 minutes with 5-minute TBS washes between each step. The sectionswere finally incubated with 0.03% H202 and 0.06% 3,3'-diaminobenzidine (BDH Chemicals, Poole, England) for 3to 5 minutes. Slides were then washed for 5 minutes inrunning tap water, counterstained with hematoxylin for 5minutes, and mounted in Canadian balsam. Endogenousperoxidase was generally visualized only in eosinophils.In some experiments, endogenous peroxidase was inhib-ited by pretreatment with 0.01% v/v H202.

The alkaline phosphatase anti-alkaline phosphatase(APAAP) technique was described in detail elsewhere.'9Briefly, sections were incubated with in turn primarymonoclonal antibodies, rabbit anti-mouse Ig (diluted 1:20in TBS supplemented with 10% normal human serum),and APAAP complexes. Each incubation step lasted 30minutes with 5-minute TBS washes between each step.The alkaline phosphatase reaction was revealed using aNew Fuchsin substrate (Sigma, St Louis, MO) (30 minuteswith continuous stirring) after adding levamisole to thesubstrate solution (final concentration 1 mmol/1) to blockendogenous alkaline phosphatase activity. Slides werethen washed for 5 minutes in running tap water, counter-stained with hematoxylin for 5 minutes, and mounted inApathy's aqueous mountant (BDH Chemicals).

In preliminary experiments the ability of the differentdeveloping reagents was compared. It was found thatPAP, ABC, strepto-ABC, and APAAP were equally effec-tive in revealing immunoreactivity for Vhp2O, BRhC3,B154.2, and B154.7 (1:100 and 1:1000 dilution of the pri-mary antibody) in LPS-stimulated PBL. In subsequent ex-periments the primary antibodies were used at 1 :100 dilu-tion to detect minimal amounts of cytokines. The percent-

IL-1 Beta and TNF Alpha in Human Tissues 891AJPNovember 1989, Vol. 135, No. 5

age of immunostained cells was determined at 400-foldmagnification on a light microscope.

Results

Immunoreactivity for IL-1 Beta and TNF Alphain Peripheral Blood Leukocytes

The presence of cells positive for IL-1 beta and TNF alphawas investigated in the peripheral blood of three normaldonors. No reactivity for IL-1 beta and TNF alpha was ob-served in freshly isolated cells. After in vitro LPS stimula-tion for 18 hours, 18% to 22% of total PBL became mark-edly immunoreactive with Vhp2O and BRhC3, suggestingthe presence of IL-1 beta. The pattern of reactivity withthe two MAbs was similar and consisted of a strong, gran-ular and/or diffuse, cytoplasmic staining (Figure 1 A). MostIL-1 beta-positive cells showed monocyte morphology,and accounted for 80% to 90% of the monocyte popula-tions. IL-1 beta reactivity was also noted in rare lympho-cyte-like cells (Figure 1A, inset) that made up less than1% of the total cells. In LPS-stimulated PBL, TNF alpha-reactive cells accounted for 5% to 7% of the total cellsand for 25% to 35% of the monocyte population. The TNFalpha staining was cytoplasmic, diffuse or dot-like (Figure1 B), and it was confined to cells with monocyte morphol-ogy. No difference was observed between B154.2 andBi 54.7 when the number of positive cells and the patternof reactivity were compared.

Immunoreactivity for IL-1 Beta and TNF Alphain Normal Lymphoid Organs

The presence of cells positive for IL-1 beta, TNF alpha, orboth was investigated on cryostat sections from normal

thymus, spleen, and lymph nodes (Table 1). Fragmentsof normal thymus were obtained from six fetuses whosegestational age ranged from 25 to 36 weeks. Thymo-cytes, cortical epithelial cells, and macrophages werenegative for IL-1 beta/TNF alpha. In the medulla, rare largedendritic cells were positive for IL-1 beta as indicated byBRhC3 and Vhp2O staining (Figure 2A).

Five histologically normal spleens were obtained frompatients with Hodgkin's disease undergoing staging lapa-rotomy. Sinusoidal macrophages of the red pulp and mac-rophages present in the periarterial lymphatic sheath(PALS) were negative for IL-1 beta and TNF alpha. Rarecells positive for TNF alpha were occasionally seen withinthe sinusoids.

Table 1. ImmunoreactivityforIL-1 Beta and TNFAlphain Cryostat Sectionsfrom NormalLymphoid Tissues

Tissue IL-1 beta TNF alpha

Thymus (N = 6)*Cortex Negative NegativeMedulla Rare large dendritic Negative

cells (Figure 2A)Spleen (N = 5)t

Red pulp Negative Rare positive cellsPALS Negative Negative

Lymph node(N = 14)t

Sinuses Negative Some cells in thesinus in 13cases (Fig. 2b)

T-dependent Some cells around Some cells in 10area HEV in 8 cases cases

B-dependent Negative Negativearea

* Six thymuses were obtained at autopsy from fetuses whose gesta-tional age ranged from 25 to 36 weeks. Cryostat sections were immuno-stained for macrophages (Dako-mac), IL-1 (BRhC3; Vhp2o), and TNF(B154.2; B154.7).

t Five spleens were surgically removed from patients with Hodgkin'sdisease for staging purpose. The spleens were not involved by the diseaseand were histologically normal.

t Fourteen superficial lymph nodes were removed for diagnostic pur-pose; histologically they were characterized by follicular, paracortical, and/or sinus hyperplasia. Eight lymph nodes were obtained from HIV-positivepatients.

Figure 1. Cytocentrifuge smears ofnormalperipheral blood leukocytes cultured withLPSfor 18hours. A: ImmunostainingforIL-1 beta with Vhp2O. Most cells with mono-cyte/macrophage morphology show diffuseand granular reactivity in the cytoplasm.Inset: A lymphocyte-like cell with markedcytoplasmic staining. B: Immunostainingfor TNF alpha with B154. 7. Some mono-cyte/macrophages show diffuse or dot (ar-rows) reactivity (PAP method, counter-stained with hematoxylin, original magni-fication X 1000).

_e' i''0

A .


Figure 2. A: Cryostat section of a normalthymus immunostained for IL-1 beta withBRhC3. A positive cell ispresent in the me-dulla (arrow) (original magnificationX400) and is characterized by abundantcytoplasm (Inset, original magnificationX 1000). B: Cryostat section of a reactivelymph node immunostainedfor TNF-alphawith B154. 7. In the sinus there are threepositive cells (original magnificationX400), whose morphology is consistentwith a macrophage origin (Inset, originalmagnification X 1000) (PAP method,counterstained with hematoxylin).

Fourteen lymph nodes with follicular, diffuse, or sinus

hyperplasia were studied, eight of which were obtained

from HIV-positive patients with persistent generalizedlymphadenopathy. Rare cells positive for IL-1 beta were

detected in the T-dependent areas of eight reactive

nodes. The positive cells had macrophage morphology

and were frequently located around high endothelial ve-

nules (HEV). Some TNF alpha-positive macrophages

were observed in 13 lymph nodes. They were singly scat-

tered in the T-dependent area, in the lymph node sinuses,

or both (Figure 2B). Germinal center macrophages and

follicular dendritic reticulum cells were consistently nega-

tive for IL-1 beta and TNF alpha.The low number of cells positive for IL-1 beta/TNF al-

pha detected in normal lymphoid tissues suggested to us

the possibility that cytokine synthesis must be induced

even in tissue macrophages. Cell suspensions from three

spleens and eight lymph nodes were cultured with LPS

for 18 hours. Cytosmears of the freshly isolated cells and

of the LPS-stimulated cells were then immunostained for

IL-1 beta and TNF alpha (Table 2). In the spleen, macro-

phages were 3% to 6% of total cells; in the lymph node

the percentage of macrophages varied (0.2% to 7% de-

pending on the pathologic condition). In unstimulatedcells, IL-1 beta and TNF alpha reactivities were noted in

the epithelioid macrophages of sarcoidosis and in a reac-

tive lymph node (TNF alpha only). After in vitro stimulationwith LPS, about 30% of spleen macrophages and 2% to

28% of lymph node macrophages became markedly pos-

itive for IL-1 beta, and 6% to 60% of lymph node macro-

phages were stained for TNF alpha. IL-1 beta/TNF alpha

reactivity was confined to cells with macrophage mor-

phology and foamy appearance (Figure 3A, B).

Immunoreactivity for IL-1 Beta and TNF Alphain Granulomatous Lymphadenitis

The reactivity for IL-1 beta and TNF alpha was investi-

gated in i1 lymph nodes involved by chronic granuloma-tous diseases (Table 3). In seven cases, the lymph node

histology and the clinical findings were indicative of tuber-


Table 2. ImmunoreactivityforIL-l Beta and TNFAlpha in Cell Suspensionsfrom Freshly Isolatedor Endotoxin-StimulatedPBL, Lymph Node, and Spleen*

Freshlyisolated LPS-stimulated

Number of % ofTissue cases Histology monocyte/macrophages IL-1 TNF IL-1 TNF

PBL 3 Normal 21-25 0 0 80-90 25-35Spleen 3 Normal 3-6 0 NEt 25-34 NEtLymph node 2 Reactive 0.3-0.4 0 0-7 4-28 8-60Lymph node 1 Sarcoidosis 7t 22 86 8 50Lymph node 3 B cell lymphoma§ 0.5-3.5 0 0 2-15 6-20Lymph node 2 Hodgkin's disease1 0.2-2.5 0 0 10-28 8-30

* Cytocentrifuge smears of freshly isolated or LPS-stimulated (20 ug/ml LPS for 18 hours) cells were immunostained with BRhC3, Vhp2O, B154.2, andB154.7. Lowest and highest percentages of monocyte-macrophages on total cells were estimated by Dako-mac immunostaining. Lowest and highestpercentages of cytokine-positive macrophages were estimated by morphology. Immunostaining with BRhC3 or Vhp2O and with B154.2 or B154.7 gavecomparable results.

t Not evaluated. A pale TNF alpha staining of doubfful interpretation was present on some macrophages prior and after LPS stimulation.t Most Dako-mac-positive cells showed epithelioid morphology. The TNF alpha staining of LPS-stimulated cells was less intense than that in freshly

isolated cells.§ The histology of the three cases of B cell lymphoma was Nodular Cb/CC, diffuse Cb, and diffuse immunoblastic.I The histology of Hodgkin's disease was mixed cellularity type.

culosis and four were indicative of sarcoidosis. A positivereaction for IL-1 beta was detected in four cases. IL-1 betareactivity was weak and was confined to small granulo-mata with a diffuse staining. A weak-to-strong TNF alphareactivity of epithelioid macrophages was noted in tencases (Figure 4). The pattern of TNF alpha staining wassimilar to that of IL-1 beta but was generally more pro-nounced.

A lymph node cell suspension was prepared in case11. As already shown (Table 2), about 80% of freshly iso-lated epithelioid cells were positive for TNF alpha and 20%exhibited a weak reactivity for IL-1 beta. As was not thecase with the other pathologic conditions, after in vitroLPS stimulation there was a gradual decrease in the per-centage of cytokine-positive epithelioid cells, as well as inthe intensity of staining.

Immunoreactivity for IL- 1 Beta and TNF Alphain Nonlymphoid Organs

The presence of TNF alpha and IL-1 beta was investi-gated on sections from autopsy and surgical specimensof several nonlymphoid organs with normal histologic ap-pearance (Table 4). Cells with IL-1 beta reactivity were notobserved in any of the tissue studied, the only exceptionbeing a weak staining in some phagocytic alveolar macro-phages of the lung. A strong immunoreactivity for TNFalpha was noted in some macrophages of the connectivefibrous stroma (Figure 5). Macrophages positive forB154.7 and B154.2 were observed in the lung interstitium,in the fibrous stroma of the breast and thyroid, in the mus-cularis propriae of the esophagus, in the dermis, and inthe interstitium of the kidney and heart. TNF alpha reactiv-ity was present in only a limited fraction of the macro-phage populations as demonstrated by Dako-mac stain-ing. Kupffer cells of the liver, nonphagocytic alveolar mac-

rophages of the lung, and intraepithelial Langerhans' cellswere consistently negative for IL-1 beta and TNF alpha.

Discussion

The presence of IL-1 beta/TNF alpha was investigated inlymphoid and nonlymphoid tissues through the use of twopairs of MAbs directed against noncross-reacting anti-gens. The specificity of the reactions is strongly sup-ported by the observation that the staining patterns witheither of the pairs were always concordant in terms ofnumber and morphology of positive cells. Furthermore,our results are extremely similar to those described in arecent report in which tissue localization of IL-1 betamRNA was investigated by in situ hybridization in C57BL/6 mice.15 This observation indicates that cytokine synthe-

AFigure&3.Cytocentrffuge smears ofLPS-stimulated cell suspen-sionsfrom spleen (A) and lymph node (B) immunostainedforIL1-beta with Vhp2O. A strong and diffuse cytoplasmic reactivityispresent in cells withfoamy macrophage appearance. In thelymph node (B), the positive cell is adjacent to a negative epi-thelioid macrophage (PAP method, counterstained with hema-toxyln, original magnification X 1000).


Table 3. ImmunoreactivityforlIL-1 Beta and TNFAlpha in Cryostat Sectionsfrom GranulomatousLymphadenitis(N= 11)

Age/sex Diagnosis* Necrosis IL-1 beta TNF alpha

23/M Tuberculosis Focal Negative Few scattered cells62/M Tuberculosis Extensive Negative Weak in epithelioid cells20/M Tuberculosis Extensive Negative Strong in epithelioid cells1 0/F Tuberculosis Extensive Weak in epithelioid cells Strong in epithelioid cells and in

scattered macrophages24/M Tuberculosis Extensive Small clusters of markedly Weak in epithelioid cells and in

positive cells scattered macrophages1 0/M Tuberculosis Focal Weak in epithelioid cells Strong in epithelioid cells and in

scattered macrophages58/M Tuberculosis Extensive Negative Moderate in epithelioid cells48/F Sarcoidosis Absent Negative Weak in some epithelioid cells and in

scattered macrophages56/M Sarcoidosis Focal Negative Weak in epithelioid cells25/M Sarcoidosis Absent Negative Weak in epithelioid cells and in few

scattered macrophages62/F Sarcoidosis Absent Weak in epithelioid cells Moderate in epithelioid cells

* The diagnosis was supported by the lymph node histology and by the clinical findings.

sis at tissue level may be investigated by either of thetechniques. However, enzyme immunocytochemistry issimpler and faster and has the advantage of visualizingthe morphology of the positive cells.

Unstimulated PBL release minimal amounts of IL-1beta/TNF alpha that are below the threshold of immu-noblot analysis.8 After LPS stimulation there is a sharp in-crease of IL-1 beta/TNF alpha synthesis as demonstratedby Northern blot and immunoblot analysis. At the singlecell level, the presence of IL-1 beta in PBL was investi-gated by indirect immunofluorescence using polyspecificsera2 and by in situ hybridization.21 By either method,unstimulated PBL were consistently negative, whereas inLPS-stimulated cultures 80% to 90% of monocytes con-tained IL-1 beta mRNA and cytoplasmic IL-1 beta. Ourfindings support and extend these previous reports. Infact, we observed that unstimulated PBL are not immuno-stained, and that after LPS stimulation 80% to 90% ofmonocytes become positive for IL-1 beta and 25% to

35% of monocytes are stained for TNF alpha. On a mor-phologic basis, immunoreactivity for TNF alpha was con-fined to monocytes, whereas the IL-1 beta staining wasalso present in rare lymphocyte-like cells whose B, T, orNK origin must be investigated further by double-stainingtechniques. Monocytes positive for TNF alpha were aboutone third of those positive for IL-1 beta. Secretion of TNFalpha is maximal after monocyte/macrophage primingwith interferon-y, GM-CSF, or both, whereas IL-1 beta re-lease does not require additional stimuli beside LPS.9 '0Thus, because we used a single agent to induce synthe-sis of TNF alpha, we cannot rule out the possibility thatthe low percentage of positive monocytes was due to theexperimental conditions.

In the thymus, we found IL-1 beta reactivity in largedendritic cells located in the medulla. It was reported thatthymic epithelial cells can secrete IL-1 beta13; moreover,rare cells positive for IL-1 beta were identified by in situhybridization in the medulla of murine thymuses.15 The

m ,, a,* < Figure 4. Cryostat section ofa lymph nodeinvolved by granulomatous lymphadenitis

7 F immunostained for TNF alpha with. B1547. A diffuse reactivity is present in

Ar. most of the epithelioid cells forming thev S* s 1 ^X granuloma (PAP method, counterstained

!4itlw>i'% with hematoxylin, original magniflcation


Table 4. ImmunoreactivityforIL-1 Beta and TNFAIpba in Cryostat Sectionsfrom NormalNonlymphoid Tissues*

Tissue Source n Macrophage localization IL-1,8 TNFa

Liver A 3 Kupffer cells - -Lung S 2 Alevolar -t -t

Interstitial - +Adenohypophysis A 3 Interstitial - +Adrenal A 3 Interstitial - +Thyroid S 2 Interstitial - +Esophagus A 1 Langerhans cells -

Interstitial - +Skin S 1 Langerhans cells -

Dermal - +Kidney A 1 Interstitial - +Heart A 1 Interstitial - +Breast S 2 Interstitial - +Brain A 1 Microglia -

* Autoptic (A) or surgical (S) specimens of fresh tissues were immunostained with BRhC3, Vhp2O, B154.2, and B154.7. The macrophage componentof the tissues was demonstrated by Dako-mac staining.

t Phagocytic alveolar macrophages containing anthracotic pigment were weakly positive for IL-1 beta and strongly positive for TNF alpha.$ TNF-positive macrophages were singly scattered in the interstitial connective tissue, and were less numerous than Dako-mac-positive macrophages.

reactivity that we observed might be ascribed to the med-ullary epithelial cells, to the interdigitating reticulum cells,which are particularly numerous in the medulla, or to both,this issue is being investigated using double-staining

Figure 5. Cryostat section of a normalheart. In the myocardial interstitium thereare three macrophages recognized by -Dako-mac (A), and a TNF-positive cell withmacrophage appearance (B) (PAP method,counterstained with hematoxylin, original : jmagnification X 1000).

techniques. In reactive lymph nodes, staining for IL-1beta/TNF alpha was noted in rare cells present in theparacortex and in the subcapsular sinus. The paracortexis the T-dependent area of the node where antigen is pre-


sented by dendritic cells/macrophages to CD4-positive Tlymphocytes. Cytokine release in this region is probablyrelated to events concerning T cell activation, prolifera-tion, or both. The subcapsular sinus is the site of entry ofthe afferent lymph. The cells positive for TNF alpha in thisregion may represent activated macrophages conveyedthrough the lymph to the sinus, or, alternatively, they mayresult from the delivery of activating signals to sinus mac-rophages.

The cell suspension study confirmed that the numberof cytokine-containing cells in the lymphoid tissues is ex-tremely low. However, it also demonstrated that lymphnode and spleen macrophages may be induced by LPSto synthetize detectable amounts of IL-1 beta/TNF alpha.As is not the case with monocytes, the ability to synthetizeIL-1 beta is restricted to 10% to 30% of tissue macro-phages. This observation is consistent with recent reportsshowing that IL-1 beta release by LPS-stimulated humanperitoneal macrophages is less than 15% of that of mono-cytes,8 and that only 43% of LPS-stimulated human alveo-lar macrophages contain IL-1 beta mRNA as demon-strated by in situ hybridization.21

Immunoreactivity for TNF alpha was particularly promi-nent in epithelioid macrophages in sections and in cellsuspension. Granuloma formation derives from lympho-cyte-macrophage interactions that are similar to those in-ducing tumoricidal macrophages in BCG-immune mice.22Moreover, it was shown that BCG-induced tumoricidalmacrophages exert their cytolytic effect through releaseof TNF alpha.23 Thus the staining for TNF alpha of epitheli-oid macrophages provides in vivo evidence that this mole-cule has a fundamental role in the expression of the gran-ulomatous reaction. It was previously demonstrated thatacquiring the "activation status" is a transitory event inmacrophage biology. In fact, cultured activated macro-phages rapidly lose tumoricidal activity even in the pres-ence of lymphokines and cannot be reactivated.24 Theseobservations are in line with the finding that cultured epi-thelioid macrophages gradually lose TNF alpha reactivityeven in the presence of LPS, and support the hypothesisthat the lymphokine pathway inducing TNF alpha synthe-sis has many similarities with that inducing tumoricidalmacrophages.9 In a recent report it was shown that theepithelioid macrophages of the sarcoid granulomata arefaintly immunoreactive for IL-1, this reactivity being morepronounced in the smaller and presumably younger gran-ulomata.14 We found that epithelioid macrophages arepoorly reactive for IL-1 beta: a clear-cut IL-1 beta stainingwas observed in only four of 11 cases of granulomatosisand in 20% of epithelioid macrophages in cell suspension.The low IL-1 beta reactivity of epithelioid cells may repre-sent an intrinsic cell property, perhaps due to terminaldifferentiation, or it may be related to regulatory mecha-nisms due to the pathologic condition.

Macrophages are widely distributed in normal tissues.We therefore investigated the IL-1 beta/TNF alpha con-tent of cells present in a variety of nonlymphoid organs.Reactivity for IL-1 beta could not be demonstrated in anyof the tissue cells, whereas interstitial macrophages em-bedded in the fibrous stroma were reactive for TNF alphain many different organs. These findings are in partialagreement with a recent report showing high levels of IL-1 beta and TNF alpha mRNA in human lymphoid and non-lymphoid organs by Northern blot analysis.25 The relation-ship between macrophages and components of the ex-tracellular matrix were extensively investigated. It wasshown that laminin and fibronectin can modulate somemacrophage functions.2627 Furthermore, it was recentlypublished that the same stimuli that induce TNF alphasynthesis, namely interferon-e and endotoxin, promotemacrophage adherence to basement membrane glyco-proteins.28 Thus, it may be speculated that TNF alpha syn-thesis is associated with macrophage penetration andtransit in the extracellular matrix of the connective tissue.

In conclusion, our results indicate that enzyme immu-nocytochemistry is a suitable and reliable technique forinvestigating cytokine production at tissue level. In the fu-ture, cytokine immunocytochemistry may be a useful toolfor further elucidating the mechanisms underlying the tis-sue lesion in immunopathology and perhaps in otherpathologic conditions.

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Acknowledgment

The authors thank F. Marziali and M. G. Saladino for excellenttechnical assistance.

immunoreactivity for il-1 beta and tnf alpha in human lymphoid

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