Workshop G Macrophages and Inflammation

Workshop G Macrophages and Inflammation

Abteilung Immunologie, Institut fur Med. Mikrobiologie und Hygiene, 7800 Freiburg, FRG

G.t Cationic antigen induced joint disease S. BATSFORD, A. MERTZ, and K. GONDOLF Negatively charged structures (proteoglycans) are found in many Jomt structures (e.g. synovial membrane, cartilage). These structures can act as binding sites for positively charged antigens, and a subsequent immune reaction (specific antibody or antigen specific T cells) can induce a chronic inflammatory reaction, as has been demonstrated in the mouse (1). We studied the influence of antigenic surface charge and size on binding to and persistence in joints. A variety of protein antigens (ov-albumin, HSA, IgG, ferritin, IgM) were chemically modified to various degrees; in addition, polymers of lysosyme were produced by crosslinking and separated by gel filtration. Wistar rats received 50 flg of each antigen directly into the knee joints, at various intervals frozen sections of whole joints were prepared and examined by immunofluorescence. Binding of antigen was highly charge dependent and occurred when the isoelectric point exceeded pH 8-9. Binding and persistance were also size dependent, as could be clearly shown with lysosyme polymers (pI 11.3), monomer (14,000) and dimer (28,000) could not be detected, but trimer (42,000) and larger polymers bound readily and persistance correlated with the degree of polymerisation. These results provide a basis for the identification of potentially damaging antigens that could induce inflammatory joint disease. 1. VAN DEN BERG, W. B., L. B. A. VAN DE PUTTE, W. A. ZWARTS, and L. A. B. JOOSTEN. 1984.

Electrical charge of the antigen determines intraarticular antigen handling and chronicity of arthritis in mice. J. Clin. Invest. 74: 1850.

'Institute for Clinical Immunology and Rheumatology, 2Institute for Clinical Pathology, 3Institute for Pharmacology of the University of Erlangen, and 4Department of Orthopedics of the Center of Rheumatic Diseases, Bad Abbach, FRG

G.2 Prostaglandin-E-2 and interleukin t~ production in mononuclear cells of patients with rheumatoid arthritis and Crohns disease W. BAUM', B. KOCH" G. R. BURMESTER', KALDEN'

J.

GIEDL2, M. REINKE 3,

J.

ZACHER" and J. R.

Previously we have shown that mononuclear cells (MNC) of patients with rheumatoid arthritis (RA) and Crohns disease (CD) produce increased amounts of prostaglandin-E-2

XXth Meeting of the Society of Immunology . 89 (PGE z) after stimulation with LPS in comparison to healthy controls (Co) (CD: n = 7, 49 ng/ ml; RA: n = 49, 23 ng/ml; Co: n = 19, 7.5 ng/ml). The present study was performed to investigate the inflammatory tissue of patients with RA and CD to produce PGE z and interleukin 113 (IL 1(3). MNC from synovial tissue (ST) from patients with RA (n = 20) and MNC from inflammatory intestinal tissue (IT) from patients with CD (n = 5) were separated by collagenase digestion and Ficoll gradient and then stimulated with LPS. PGE z and IL 113 were measured in RIA systems. PGE z was found to be significantly increased in the ST fraction of RA (300 ± 19 ng/ml), but less increased in IT -MNC from patients with CD (22.4 ± 17.5 ng/ml). IL 113 production was found to be elevated in the blood MNC fraction up to 8 ng/ml after LPS stimulation in some patients (X RA: 4.9 ± 1.7 ng/ml; X CD: 4.6 ± 2.8 ng/ ml), whereas the mean production of Co was 3.5 ± 1.5 ng/m!. In comparison to blood MNC, tissue MNC spontaneously released increased amounts of IL 113 (Blood X = 1.0--2.1 ng/ml; Tissue X = 3.0--3.1 ng/ml). From our data it could be seen, that PGE z release in RA-ST was 10 to 20-fold greater than that of CD-IT, while IL 113 production was at a normal range in the inflammatory tissues from both diseases.

Supported by BMFT.

Institute of Immunology and Genetics, German Cancer Research Center, D-6900 Heidelberg, FRG

G.3 Involvement of separate signal pathways in the stimulation of peritoneal macrophages by bacteriallipopolysaccharides. Analysis of the effects of incomplete LPS structures B. BENNINGHOFF, W. DROGE, and V. LEHMANN Bacterial lipopolysaccharides and several incomplete LPS structures from Salmonella typhimurium which lack either the lauric and myristic acid residues or the carbohydrate moiety (i.e. lipid A) stimulate macrophages to convert arginine into ornithine, a substance which was shown to be important for T cell activation. Two different incomplete LPS structures which lack carbohydrate and lauric and myristic acid (i.e. the lipid A precursors IA and IE) fail to stimulate ornithine production. The stimulation of ornithine production by LPS is inhibited by the cyclooxygenase inhibitor indomethacin and reconstituted by exogenous PGE z or cholera toxin, i.e., two substances which are known to raise the intracellular concentration of cyclic AMP. Moreover, the LPS-mediated effect is augmented in a synergistic manner by PGE z, by cholera toxin and by dibutyryl-cyclic AMP. The lipid A precursors IA and IE which fail to stimulate ornithine production are, nevertheless, capable of stimulating PGE z synthesis. The results of our experiments suggest that the LPS-induced production of PGE z and the PGErinduced increase of the intracellular cAMP concentration are essential elements of an autoregulatory loop that controls the LPS-induced stimulation of the ornithine production. Stimulation of the autoregulatory loop is necessary but not sufficient for this effect, thus revealing the requirement for an additional signa!. This additional signal requires the lipid A precursor structure to be substituted either by the carbohydrate region or by the lauric and myristic acid residues.

90 . XXth Meeting of the Society of Immunology Medizinische Klinik, Hugstetter Str. 55, 7800 Freiburg, FRG

GA Macrophage colony-stimulating factor is required for monocyte survival and acts as a cofactor for their terminal differentiation to macrophages in vitro W. BRUGGER, A. REHM, and R. ANDREESEN Functional competence as well as phenotype heterogeneity of the macrophage (M
Institute of Clinical Immunology and Rheumatology, Institute of Biochemistry II, University of Erlangen-Niirnberg, Krankenhausstr. 12, 8520 Erlangen, FRG

G.5 Activation of blood monocytes and synovial macrophages by recombinant interferon-y (IFN-y) and tumor necrosis factor-a (TNF-a) in patients with rheumatoid arthritis (RA) G. R. BURMESTER, G. HAHN, W. KERSTEN, E. PLATZER, and

J.

R. KALDEN

Rheumatoid arthritis is an inflammatory disorder characterized by a highly activated state of blood monocytes and synovial macrophages. In order to study possible mechanisms of monocyte/macrophage activation, rheumatoid and normal control monocytes as well as RA synovial macrophages were cultured in the presence of recombinant IFN-y and/or TNF-a. Cell activation was determined by the release of prostaglandin E2 (PGE 2), neopterins and interleukin 1 (IL 1) upon stimulation. Immediately after separation by Nycodenz-gradients, RA monocytes already at baseline level showed a high secretion of PGEz, neopterins and IL 1 compared to normal donors - which could even be enhanced by the addition of IFN-y and TNF-a with synergistic effects. Interestingly, TNF-a was able to induce extremely high levels of neopterins in both blood monocytes and tissue macrophages in RA patients, while normal control monocytes could not be stimulated to release neopterins by this mediator. After

XXth Meeting of the Society of Immunology . 91 therapeutic leukapheresis performed in two patients with severe RA, the properties of blood monocytes changed dramatically from a highly activated state to normal levels of PGE2 and neopterin secretion. These data indicate that activated monocytes/macrophages significantly participate in the rheumatoid inflammation and that the therapeutical benefit of leukapheresis may be explained by the removal of activated monocytes.

Forschungsinstitut Borstel, Institut fUr Experimentelle Medizin und Biologie, D-2061 Borstel, FRG

G.6 Inhibitors of lipoxygenases suppress mitogenic activity of endotoxins in murine spleen cell cultures E. ELEKES, E. TH. RIETSCHEL, and U. F. SCHADE Mitogenic activation of B lymphocytes has been described to require the presence of macrophages. Since several aspects of macrophage stimulation depend on the activity of cellular lipoxygenases, it seemed of interest to study the involvement of this enzymatic pathway in the endotoxin (LPS)-induced mitogenicity in murine spleen cell cultures. The mitogenic effect of LPS (5. abortus equi) was determined by the 3H-thymidine incorporation assay. Selectivity of the individual inhibitors was determined by measuring their effects on the synthesis of prostaglandins and leukotriene C 4 in zymosan-stimulated mouse peritoneal macrophages. BW 7SSG, a known blocker of the cyclooxygenase and the lipoxygenase pathway, depressed LPS-induced spleen cell proliferation markedly and in a dose-dependent way. However, indomethacin, a cyclooxygenase inhibitor did not effectively interfere with thymidine uptake at concentrations specifically blocking prostaglandin synthesis in macrophages. The selective lipoxygenase inhibitor 210-610 (kindly provided by Sandoz, Basel) was found to block LTC. production in macrophages (ca. 50 % inhibition, PGE2 synthesis: 146 % of controls at 3 X 10-6 M) and inhibited spleen cell proliferation (3 X 10- 6 M: 40 %). The same correlation between blockade of LTC4 synthesis and suppression of spleen cell proliferation was observed when the lipoxygenase inhibitor 208-199 (Sandoz, Basel) was tested. These results indicate that lipoxygenases could play a role in LPS-induced mitogenicity in murine spleen cells. Supported by the Deutsche Forschungsgemeinschaft (Scha 402/1-2).

DRK-Blutspendezentrale and Abt. Transfusionsmedizin der Universitat, Oberer Eselsberg 10, D-7900 UIm, FRG

G.7 Lipoproteins suppress endotoxin-induced monokine-release W. A. FLEGEL and H.

NORTHOFF

Monocytes release lymphokines [interleukin 1 (IL 1), tumor necrosis factor (TNF), interleukin 6 (IL6)] upon stimulation with endotoxin [lipopolysaccharide (LPS)]. They mediate a substantial part of the LPS-toxicity in vivo. We have shown earlier that normal human sera can neutralize endotoxin, rendering LPS unable to induce IL 1 release by human monocytes. This LPS neutralizing capacity resides exclusively in the lipoprotein fraction. Here, we report

92 . XXth Meeting of the Society of Immunology experiments where both low density lipoprotein (LDL) and high density lipoprotein (HDL) have a clear-cut neutralizing effect - other than might be expected from the literature. LPS neutralizing capacity is shown for several species of lipopolysaccharides, for natural and a synthetic lipid A. We could exclude that nutritive lipids play any important role, since visibly lipemic sera had no enhanced capacity for neutralizing endotoxin. Naturally occurring antibodies against endotoxin could be excluded to have a discernible effect in our system. Further, neutralization is not due to complement. In conclusion, interaction of endotoxin with human lipoproteins reduces the ability of LPS to induce IL 1 release by human monocytes. LPS neutralizing capacity is shown for HDL and LDL. Other components of normal human serum have no comparable effect at all. We think that lipoproteins may also represent an LPS detoxifying principle in man.

Inst. f. Allg. und Exper. Pathologie, Universitat Wien, Austria

G.8 Phagocytosis via a sialic acid binding receptor on rat macrophages: conditions necessary for triggering the phagocytic response A. GESSEL, T. PERNERSTORFER, H. NEMET, G. BOLTZ-NITULESCU, and O. FORSTER Previous studies in several laboratories have shown that macrophages recognize sialic acid containing structures Immunol. 128: 1205 (1982), J. Leukocyte BioI. 37: 289 (1985», obviously via a specific receptor in their plasma membrane (Immunology 51: 177 (1984), Molec. Immunol. 23: 1167 (1986». Resident rat alveolar macrophages in our hands could bind but not ingest ganglioside coated sheep erythrocytes (EG) through this receptor. Recent studies in our laboratory have shown that bone marrow-derived macrophages (BMDM) as well as cultured alveolar macrophages from rats were able to phagocytize EG. The conditions which induce this functional activity were studied mainly in BMDM. It was shown that IFN-a and IFN-~ markedly enhanced the phagocytic activity for EG. This effect was dose-dependent and could be abrogated by an antiserum against IFN-a!~. IFN-y either had no effect or reduced phagocytosis of EG, probably by reducing the expression of the sialic acid receptor (SAR) on macrophages. TNF-a led also to a slight decrease of EG-ingestion at high concentrations. It appears that upon certain activation signals, macrophages can be induced to phagocytize particles which are recognized by their content of sialic acid terminated glycoconjugates. This may be of importance in the defence against bacteria which are able to produce sialic acid containing capsules.

a.

Supported by Austrian Science Research Funds, Proj. Nr. 5056.

Abteilung fiir Immunologie, Kreuzbergring 57, D-3400 Gottingen, FRG

G.9 Accessory cells differentiated from murine bone marrow in a serum-free liquid culture system R. K. H. GIESELER, and J. H. PETERS Recently, we have shown that accessory cells (AC) can be obtained from cultured monocytes (MO) at a stage between MO and macrophages (Mph), both in the human (1) and murine

XXth Meeting of the Society of Immunology . 93 system (unpublished). Here we report that AC can be differentiated from bone marrow (BM) precursors under serum-free conditions. BM cells were flushed from femurs and tibias of 6-week-old Balb/c mice. Single cell suspensions were allowed to adhere for 3 h either on hydrophilic or hydrophobic Petri dishes. Adherent and non-adherent (NAd) cells were cultured again on hydrophilic or hydrophobic plastic at 5 x 105 cells/200 Ill. Different combinations of culture media were prepared from: 80% RPMI 1640/20% medium 199 (80120) and 80120 plus 10 % basal medium supplement (Biochrom) (80/20/BMS); L929 cellconditioned medium (L-CM), containing macrophage growth factor (MGF), was added for different periods of time. Cultures were maintained for 8 days. BM accessory activity was reflected by periodate-induced proliferation of added lymphocytes. A subpopulation of BM cells could be expanded by the MGF stimulus leading to cells of high accessory activity, which were by a factor of 140 more potent (d 8) than the starting population. These cells were found in the NAd fraction obtained and cultured on hydrophilic plastic in 80/20/BMS supplemented with 5 % L-CM for the entire culture period. They were weakly positive for non specific esterase (NSE), negative for Fc receptors (FcR), and negative for latex particle phagocytosis. Differentiation of these cells revealed small, round cells at dO, irregular cells with protrusions at d 2, veiled cells at d 3, and dendritiform veiled cells of stretched shape at d S-8. The number of AC obtained correlated with the duration of MGF presence, leveling off at approximately d 6--7. As these cells did not express Mph phenotype (NSE, FcR, phagocytosis), in contrast to serum supplemented cultures, we conclude that serum starvation has prevented the cells from differentiating into Mph; instead, they persisted at a stage preceeding terminal Mph differentiation, representing highly active AC. 1. PETERS, J. H., S. RUHL, and D. FRIEDRICHS. 1987. Veiled accessory cells deduced from monocytes. Immunobiol. 176: 154-166. Supported by DFG, SFB 236.

Division of Molecular Pharmacology, Medical School, Hannover, FRG

G.lO Release of tumor-necrosis-factor-alpha (TNF-a) and interleukin 1 (IL 1-~) after phorbol erster-induced differentiation of U937 cells R. HASS, G. LONNEMANN, L. KOHLER, M. GOPPELT-STRUBE, and K. RESCH Cells of the human histiocytic lymphoma cell-line U937 failed to release detectable amounts of TNF-a and IL 1-~. However, when U937 cells were induced to differentiate along the monocyte/macrophage lineage by incubation with 12-0-tetradecanoylphorbol-13-acetate (TPA), both IL 1-~ and TNF-a release into the culture medium as measured by radioimmunoassay were markedly increased. TNF-a was released from U937 cells after 30 min treatment with TPA, this level increased in a time-dependent manner and was maximal by 24 h. During phorbol ester-induced differentiation of U937 cells, marked morphological and functional changes were observed including increasing adherence and inhibition of proliferation. Thus, by 24 h after TPA treatment, U937 cells ceased to divide and simultaneously, these cells began to release IL 1-~ reaching a maximum after 72 h. These data demonstrate that U937 cells acquire the capacity to release significant amounts of TNF-a and IL 1-~ after phorbol esterinduced differentiation into macrophage-like cells; however, the TNF-a is released immediately after TPA-stimulation, suggesting the presence of preformed TNF-a in U937 cells, whereas IL 1-~ release is mediated by and induced protein expression.

94 . XXth Meeting of the Society of Immunology Fraunhofer Institute for Toxicology, Dept. of Immunology, 3000 Hannover 61, FRG

G.tt Splenic macrophage precursor cells from Leishmania donovani infected mice as cytotoxic effectors against pro- and amastigotes S. HOCKERTZ, and M.-L. LOHMANN-MATTHES The most important effectors of natural- as well as lymphokine-mediated cytotoxicity against microbicidal and fungal targets and protozoa such as Leishmania donovani are represented by cells of the monocyte-macrophage lineage. We recently described the bone marrow-derived macrophage precursor which is able to spontaneously and extracellularly kill protozoa of the genus Leishmania. These nonadherent, nonphagocytic macrophage precursor cells are present in the spleen of healthy mice only in a small quantity; however, high numbers have been isolated from the spleen of L. donovani infected mice. Macrophage precursors from spleen of diseased animals are able to kill spontaneously the promastigote as well as the amastigote form of L. donovani. The mechanism of the spontaneous leishmanicidal activity of macrophage precursor cells derived from spleens of L. donovani infected mice was investigated. This effector function could be defined in part as an antibody-dependent cellular cytotoxicity (ADCC). In addition we assessed the role of CSF I containing L-cell conditioned supernatant at the leishmanicidal activity of these immature cells of the macrophage lineage. For that purpose, nonadherent cells from healthy mice were cocultivated with this CSF I containing medium for four days. These in vitro proliferated macrophage precursor cells from untreated mice showed an increased leishmanicidal activity. Thereby we established a further activation mechanism for splenic macrophage precursor cells responsible for the observed killing of L. donovani pro- and amastigotes.

Institut fUr Immunbiologie der Universitat, D-7800 Freiburg, FRG

G.t2 Tumor cytotoxicity involving TNF induced in bone marrowderived macrophages by two lipopeptide analogues of bacterial lipoprotein P. HOFFMANN and W. G. BESSLER The lipoprotein from E. coli, its N-terminallipopeptide structure, and several analogues prepared by chemical synthesis constitute potent B lymphocyte (1) and macrophage (2) activators. Pam3Cys-Ala-Gly and Pam3Cys-Ser-(Lys)4 (S-2,3-bis(palmitoyloxy)-(2RS)propyl)-N-palmitoyl-(R)-cysteinyl-alanyl-glycine and -cysteinyl-seryl-tetralysine) were also able to induce tumor cytotoxicity in bone marrow-derived macrophages, as tested in a 3H_ thymidine release assay on the fibroblast cell line L929. After stimulation with the lipopeptides, macrophages exhibited a direct cytotoxicity most pronounced at days 8-10 of culture. Specific cytolysis of 60-70 % was obtained using activator concentrations between 10 and 100 J.lg/ml. The effect of the analogues was comparable to LPS. Moreover, the supernatants of lipopeptide-stimulated bone marrow-derived macrophages were tested for the presence of cytotoxic factors. In the in vitro test for tumor necrosis factor (TNF) they exerted a cytotoxic effect on the L929 cell line and proved to contain up to 100 cytotoxic units/ml. The cytotoxic activity in the supernatants could be inhibited by addition of an anti-muTNF antiserum. Our

XXth Meeting of the Society of Immunology . 95 results show that TNF is involved derived macrophages.

III

lipopeptide-induced cytotoxicity in bone marrow-

1. BESSLER, W., M. Cox, A. LEX, B. SUHR, K. H. WIESMULLER, and G. JUNG. 1985.

J.Immunol. 135: 1900-1905. 2. HOFFMANN, P., S. HEINLE, U. F. SCHADE, H. LOPPNOW, A. J. ULMER, H.-D. FLAD, G.JUNG, and W. G. BESSLER. 1988. Immunobiol. 177: 158. Supported by the Deutsche Forschungsgemeinschaft.

Forschungsinstitut Borstel, Department of Immunology and Cell Biology, Parkallee 22, D-2061 Borstel, FRG

G.B Phagocytosis of mycobacteria by human monocytes: relationship between monocytic chemiluminescence (CL) and antimycobacterial activity L. HUBNER, M. ERNST, and H.-D. FLAD

The interaction between intracellular mycobacteria and mononuclear phagocytes is not fully understood. We investigated the killing rate of Mycobacterium leprae (M. leprae), M. «lufu», and M. tuberculosis (Middelburg) and the phagocytosis associated formation of activated oxygen species by human monocytes. The phagocytosis associated release of activated species as measured by luminol dependent CL was lowest with M. leprae and highest with M. tuberculosis but was elevated in either case following pretreatment of monocytes with interferon-y (IFN-y). Our finding that the zymosan induced luminol dependent monocyte CL was rapidly reduced after addition of M. leprae led us to the hypothesis that M. leprae could inhibit phagocytic myeloperoxidase (MPO). Indeed, as with the known MPO-inhibitor NaN}, addition of M.leprae but not of M. tuberculosis to zymosan phagocytosing monocytes led to an increased CL response in the lucigenin dependent CL system. In contrast, the quantity of mycobacterial uptake by phagocytes was lowest with M.leprae. We conclude from our data that 1. the three different species of mycobacteria exhibit differential susceptibility to intracellular killing by human monocytes. 2. Pretreatment of monocytes with IFN-y increased (or induced, as was the case with M. tuberculosis) the monocytic anti-mycobacterial activity to all three mycobacterial species as well as the phagocytosis associated CL responses. 3. The release of activated oxygen species from monocytes is not directly correlated to the subsequent intracellular killing. 4. M. leprae may partially escape from damage by oxygen radicals by inhibiting the monocytic MPO.

Division of Molecular Pharmacology, Medical School, Hannover, FRG

G.14 Induction of prostaglandin secretion during differentiation of the histiocytic cell line U937 L. KOHLER, R. HAss, M. HADAM, M. GOPPELT-STRUBE, K. WESSEL, V. KAEVER, and K. RESCH

The human histiocytic cell line U937 can be induced to differentiate into macrophage-like cells following 3 days incubation with 5 X 10-9 M 12-0-tetradecanoylphorbol-13-acetate

96 . XXth Meeting of the Society of Immunology (TPA). During the differentiation process the cells ceased to divide as detected by lH_ thymidine incorporation; and simultaneously the percentage of cells expressing transferrin receptors as detected by fluorescence-activated-cell-sorter analysis was reduced by 70 %. After treatment with TPA, the undifferentiated U937 cells underwent several morphological changes. The original round cell growing in suspension formed cell clusters, extended pseudopodia and adhered to the plastic dishes. Beside the morphological changes, an induction of the lysosomal enzyme a-naphtyl-esterase was also observed. An important property of macrophage-like cells is the capacity to produce eicosonoids. The undifferentiated U937 cells secreted only small amounts of prostaglandin E2 (PGE2) and thromboxane (TxB2) when stimulated with exogenous arachidonic acid. In contrast TPA-differentiated cells produced 20-30-fold more prostaglandins. During the differentiation process, the increase of prostaglandin secretion became significant after 18 h and reached its maximum after 48 h. In order to obtain further insight into the underlying mechanism of this difference, we examined the enzymes involved in regulating the availability of the precursor of prostaglandin synthesis arachidonic acid. Phospholipase A2, which cleaved fatty acids from phospholipids, was induced 2-fold in TPA-differentiated U937 cells, whereas lysophosphatide acyltransferase reacylating fatty acids into phospholipids remained unchanged. No leukotrienes could be detected in either undifferentiated or TPA-differentiated U937 cells. These results suggest that the capacity to produce appreciable amounts of prostaglandins is acquired during differentiation into macrophage-like cells possibly by induction of cyclooxygenase. The ability to secrete leukotrienes is possibly a later event in differentiation, not reached under the conditions of TPA-differentiation in this cell line.

Institute of Immunology, University of Vienna, Borschkeg. 8a, 1090 Vienna, '1st Department of Medicine, University of Vienna, 1090 Vienna, Austria

G.15 Altered expression of surface antigens on human granulocytes, activated in vitro or in vivo D. MAURER, G. F. FISCHER, W. HINTERBERGER', and W. KNAPP We show that in vitro activation of human granulocytes leads to an altered expression of distinct surface antigens. These changes are most dramatically seen upon stimulation of granulocytes by the phorbol ester PMA. Activation with this compound, e.g., is followed by an enhanced expression of complement receptors, while certain Fc receptors are diminished on the cell surface. Similar, but less pronounced alterations in surface antigens are observed upon granulocyte activation by therapeutically relevant cytokines as, e.g., recombinant granulocytemonocyte colony stimulating factor (rGM-CSF) or TNF-alpha. We became particularly interested in studying whether the observed changes in surface antigen expression upon in vitro activation would have a correlate on in vivo activated granulocytes. For this purpose, we monitored the in vivo effects of rGM-CSF during therapy. Granulocytes of patients being treated with continuous infusion of rGM-CSF were stained with monoclonal antibodies and analyzed by flow cytometry. We found an impressive increase in CR1 and CR3 expression and a significant decrease in the amount of CD16 antigen on granulocytes of the rGM-CSF treated patient. These changes: 1) correlated well with our in vitro data, 2) took place extremely rapidly after therapy onset (within hours) and 3) occurred even before any alterations in differential blood count could be observed. We conclude that expression of granulocyte differentiation markers change upon activation in vitro and in vivo. Furthermore, analysis of this activation associated changes in antigen expression could be useful parameters in monitoring the in vivo effects of rGM-CSF.

XXth Meeting of the Society of Immunology . 97 IInstitute of Immunology, 2Department of Internal Medicine, Heidelberg, FRG

G.16 Unilateral nephritic effect of cobra venom factor (CVF) targeted into the right kidney of experimental rats R. METZI, M. ZEIER2, M. RAGHUNATIlt, J. NITSCHt, E. RITZ2, and E. W. RAUTERBERG 1 Deposits of complement (C) components in histologically altered glomeruli are a common feature in most types of human glomerulonephritis. The pathogenetic role of the C-system in inflammation and formation of lesions is, however, predominantly unclear. To elucidate the effect of a local C-activation, we developed a new experimental antibody-independent nephritic model: varying doses of CVF purified to homogeneity from crude venom of Naja naja khaoutia were injected into the right kidney of male Wistar rats via the suprarenal artery. CVF is known as a potent activator of the alternative C-pathway. Localization of CVF in the glomeruli was improved by preceding bolus injection of concanavalin A (Con A) into the kidney. Control rats received saline or Con A alone, respectively. Animals were sacrificed 2 h or 3 days after injection. Cryostat sections of the left (untreated) and right kidney were subjected to histological (HE and PAS stain) and immunohistochemical stainings using monoclonal antibodies (Mabs) against rat granulocytes or macrophages. Monocytes were furthermore visualized by demonstrating their alpha-naphthyl-butyrate-esterase activity (NBE). The following parameters were determined in both kidneys: diameter of glomeruli, number of nuclei, of NBE- and of Mab-positive cells per glomerulus. At a dose of 50 Ilg, CVF alone caused no measurable effects. In contrast, 50 Ilg CVF, preceded by 500 Ilg ConA, resulted in significant enlargement of glomeruli of the treated kidney after 2 h as compared to the untreated kidney (p<0.001). This unilateral effect of CVF/ConA treatment was confirmed regarding other parameters: the number of nuclei per glomerulus was distinctly increased as well as the number of cells identified by Mabs against either granulocytes or monocytes. The alterations remained visible for (at least) 3 days: 50 Ilg CVF preceded by 100 Ilg Con A lead to significant unilateral swelling of the glomerular tuft and invasion of granulocytes in the treated kidney. Administration of Con A (200 Ilg) alone or saline gave negative results. In vivo binding of Con A and CVF to glomerular sites was confirmed by immunohistochemical staining. Our findings indicate that: 1) CVF can elicit nephritic effects in the absence of antibodies, and 2) this model is suitable to further elucidate the role of activated C-products in the induction of glomerular lesions.

Department of Immunology, Gottingen; FRG

G.17 Adenosine as a regulatory molecule on the ,differentiation of monocyte-derived dendritic cells (m-DC) H. M. NAJAR, A. BRU-CAPDEVILLE, and J. H. PETERS Our initial finding had revealed that monocytes, on their way to becoming macrophages (McII), pass through a transient stage of high accessory activity, when cultured in medium containing 20 % human serum. This transient stage can be prolonged by omission of serum, leading to monocyte-derived dendritic cells (m-DC). We have investigated the signals responsible for Mell and m-DC differentiation. Incubation of monocytes with agents recognized to

98 . XXth Meeting of the Society of Immunology promote increase in intracellular cAMP (i.e. epinephrine, NaF, dbcAMP, cAMP, theophylline and euphylline) caused an increase in their accessory function as measured by the potency of m-DC to support lymphocyte proliferation. These agents were able to prevent Mell differentiation (caused by serum) and to keep the cells in the m-DC state. This differentiation state was accompanied by formation of veils and dendritiform elongations. The treated cells were loose\y adherent and lacked granula. Unlike MeII, m-DC were weakly positive or negative for non-specific esterase and Fc receptor. Fc receptor dependent and independent phagocytosis were reduced in m-DC in comparison with monocytes or Mell. This provides evidence that increase in intracellular cAMP causes the cells to stay in their accessory state. Furthermore, we have also shown that addition of adenosine to monocytes, activates the adenosine receptor (Az) on the cells which stimulates adenylate cyclase and increases cAMP, and hence the cells obtain m-DC criteria. Adenosine receptor is located on the outside of the cell membrane and reacts with the ribose moiety of adenosine. M-DC differentiation was also enhanced by other adenine nucleotides such as AMP, ADP, and ATP. It is therefore probable that these nucleotides are converted to adenosine in culture, and their effects are mediated via the adenosine receptor. It seems that adenosine acts as a regulator of cellular function and differentiation.

Institute for Medical Microbiology, Johannes Gutenberg-University, Hochhaus am Augustusplatz, 6500 Mainz, FRG

G.18 In vivo stimulated peritoneal macrophages induce bactericidal activity in resident bone marrow-derived macrophages via an IFNy-like monokine J.

u. PAULY, H. G. FISCHER, and U. HADDING

Peritoneal exudate cells obtained 8 days after serial immunization of C57BLl6 mice with Listeria monocytogenes were enriched for macrophages (MeII) by adherence followed by repeated washing steps. The population consisted of more than 98 % of Mell as determined by phagocytosis of latex beads, staining for nonspecific esterase and MAC-1 expression. After removal of possible contaminating T cells by treatment with anti-Thy-t antibody and complement, the cells did not respond to stimulation with concanavalin A. Cultivation of these purified Mell under serum-free conditions yields a supernatant, which exhibits macrophageactivating factor (MAF) activity. This was shown in functionally resident bone marrowderived Mell via the induction of 1) the capacity to restrict the intracellular growth of Listeria monocytogenes, 2) Ia expression and 3) antigen presentation function towards an insulinspecific murine helper T cell clone. MAF activity was found to be optimal 48 h after starting the Mell cultures and could be detected up to 6 days, while the medium was changed every 2 days. Physico- and immunochemical characterization of the active fraction (M.W. > 30,000 D, sensitive to pH 2-treatment, stable at 56°C, detectable by a monoclonal anti-IFNy antibody) suggests that the activity could be contributed to IFNy produced by the peritoneal Mell. Therefore, we postulate IFNy to act as a stimulatory signal within a MeII-MeII interaction triggering Mell effector functions. Supported by BMFT grant No. PBE/t-03 87276.

XXth Meeting of the Society of Immunology . 99 Fraunhofer-Institut fur Toxikologie und Aerosolforschung, Nikolai-Fuchs-Str. 1, 3000 Hannover 61, FRG

G.19 Defense of murine bone marrow radiation chimeras against Listeria monocytogenes in the early phase after transplantation

J.

ROESLER, E. GROTIRUP, and M.-L. LOHMANN-MATTHES

In the early phase after clinical or experimental bone marrow transplantation, the recipient is unable to respond specifically to a given antigen, due to a lack of functional T cells. In contrast, we could show that efficient function of phagocytes (and natural killer cells) can reappear quickly and mount defense against invading microorganisms. Already on day S and day 10 after transfer of T cell-depleted CS7BLl6 bone marrow cells into lethally irradiated DBA/2 mice, we could find cells with normal or in some organs even increased potency to phagocyte sheep erythrocytes, to produce reactive oxygen intermediates, to kill P81S or YAC-l tumor cells and to produce tumor necrosis factor. As compared to controls the chimeras showed a surprisingly high increase of CFU-reduction (10 3 vs. 105) in spleen and liver after intravenous infection with the bacteria Listeria monocytogenes (L.m.) on day 10. If L.m. was incubated with spleen cells of the chimeras in vitro killing of the bacteria was performed by nylon wool adherent but not by immature cells. After Percoll density centrifugation of splenocytes, the CFU reducing capacity of the cell fraction enriched with macrophages exceeded that of the fraction enriched with polymorphonucleated cells. Moreover, in vivo clearance of L.m. was in part destroyed by administration of dextran sulfate, demonstrating efficient antilisterial macrophage activity. We conclude that phagocytes especially pre activated macrophages can in part compensate for defective T cell-dependent defense mechanisms after bone marrow transplantation.

Forschungsinstitut Borstel, Institut fur Experimentelle Medizin und Biologie, D-2061 Borstel, FRG

G.20 Possible role of 13-hydroxylinoleic acid in the activation of macrophages by endotoxin U. F. SCHADE, I. BURMEISTER', H. LODE, R. ENGEL, and E. TH. RIETSCHEL It has been previously found that inhibitors of lipoxygenases interfere with the stimulation of macrophages by endotoxins (secretion of interleukin 1, enhanced phagocytosis). It is, therefore, possible that products of this metabolic pathway are involved in the activation of macrophages. In order to obtain information on the nature of the lipoxygenase product in question, we tested the secretory leukotrienes C 4 and B4 (L TC 4, LTB 4) and 13-hydroxylinoleic acid (13-hydroxyoctadecadienoic acid, 13-HODD, a potential mediator of inflammation) with regard to their capacity to stimulate phagocytic properties of macrophages in comparison to endotoxin. LTC4 and LTB4 were not able to stimulate the phagocytic potential of mouse peritoneal macrophages. 13-HODD (3-30 X 10- 9 M), however, increased the uptake of zymosan particles to the same degree as LPS (10-100 ng/ml). Interestingly, in hydrolysates of mouse peritoneal macrophages treated with LPS in vitro, we detected increased amounts of 13HODD (4-8 times more than in unstimulated cells). A careful analysis of the stereochemical properties of the material revealed its enzymatic origin. 13-HODD could not be detected in

100 . XXth Meeting of the Society of Immunology supernatants or cellular extracts which were not subjected to hydrolysis. This indicated that 13-HODD was covalently bound to cellular components. These results suggest an involvement of 13-HODD in the stimulation of macrophages by LPS. Supported by the Deutsche Forschungsgemeinschaft (Scha 402/1-2). "Recipient of a scholarship of the Kultusminister of Schleswig-Holstein (3156.45-7-2).

Med. Microbiology and Immunology, AG Infektabwehr, Ruhr Universitat Bochum, FRG

G.21 Metabolism of leukotriene B4 in human lung macrophages W. SCHONFELD, R. HILGER,

J.

KNOLLER, and W. KONIG

We recently analyzed leukotriene generation in human lung macrophages from solid lung tissue as well as from bronchoalveolar lavage in physiological and pathophysiological conditions. The formation of unpolar metabolites of LTB 4, a pathway associated to human macrophages derived from tonsillar tissue and lung tissue was observed. By chromatographic analysis (HPLC, radioactive HPLC, radioactive thin layer chromatography), it could be demonstrated that the main metabolite of LTB4 is the dihydro-LTB 4 (5,12-dihydroxyeicosatrienoic acid), which is formed by reduction of one of the conjugated double bonds. In bronchoalveolar lavage, which mainly consists of macrophages, a rapid conversion almost exclusively into dihydro-LTB 4 and two further products which are not yet identified could be monitored whereas no Ol-oxidated products are detected in pure macrophage suspensions. LTB 4-conversion was pH- and temperature-dependent (optimum at pH 6.5 at 37"C). The metabolites showed no crossreactivity with thei LTB 4-antiserum and a reduction of the chemotactic activity was observed after incubation of LTB4 with macrophages; these data indicate a reduced biological potency of dihydro-LTB4' Incubation of dihydro-LTB4 with granulocytes leads to the formation of polar substances probably Ol-oxidated products of dihydro- LTB 4; therefore up to seven metabolites can be detected by radioactive HPLC after incubation with 3H -LTB4 of bronchoalveolar cells consisting of macrophages and granulocytes. Our data provide evidence that in the lung and other macrophage-rich tissues the main pathway of LTB4 inactivation is the formation of dihydro-LTB 4. The role of these metabolites in various disease processes of the lung is currently being studied.

Med. Institute of Environmental Hygiene, D-4000 Dusseldorf 1, FRG

G.22 High- and low-responder mouse strains with respect to silicainduced fibrosis M. STARK, S. ZAIDI, B. HILSCHER, W. HILSCHER, and E. GLEICHMANN Fibrosis consists of an accumulation of fibroblasts and their extracellular product collagen. The pathogenesis of fibrosis is incompletely understood. Using quartz-induced fibrosis as a model, we tried to identify high- and low-responder mouse strains because comparisons of such strains might improve our insight into the fibrotic process. Quartz (2.5 mg of DQ12) was injected into one hind footpad of mice, and the popliteal lymph nodes (PLNs) were removed on days 40, 90, and 180, respectively. The draining PLN showed a similar progressive enlargement (up to a factor of 200) in all 6 mouse strains tested, and in strains BALB/c and

XXth Meeting of the Society of Immunology· 101 DBAI2, PLN enlargement was virtually identical. However, when the fibrotic areas (quartztypical areas, QTA) in the draining PLN were determined by morphometry, marked strain differences were found: on day 180, 11 % of the total PLN area was occupied by QTA in strain BALB/c, but only 0.3 % in strain DBAI2 (both H-2 d ). A comparison of BALB/c nu/nu and +Inu mice showed that quartz-induced fibrosis does not require T cells. These findings fit the notion that quartz (Si02) is not an antigen. In conclusion, we were able to separate the lymphoproliferative effect of quartz from its fibrogenic effect and showed that MHC molecules and T cells are not involved in quartz-induced fibrosis. This leaves macrophages, which are activated by silica and known to produce fibrogenic cytokines, and fibroblasts, which respond to these cytokines, as the most likely candidate cells expressing the genetic difference described.

Abteilung Immunologie, Institut fur Med. Mikrobiologie und Hygiene der Universitat, 7800 Freiburg i. Br., and Institut fur Med. Mikrobiologie, Med. Hochschule, 3000 Hannover, FRG

G.23 Monocyte involvement in passive Heymann nephritis A. VOGT, M. HARA, D. BITTER-SUERMANN, and S. BATSFORD Previous studies have demonstrated a role for complement but not for polymorphonuclear granulocytes (PMN) in passive Heymann nephritis in the rat (1). We extended these studies and identified monocytes as another important mediator. Passive Heymann nephritis was induced in Wistar rats by intravenous injection of sheep anti-rat Fx1A. Four groups were studied: (A) rats given normal sheep IgG (nephritic controls), (B) rats given sheep anti-rat PMN IgG (PMN depleted), (C) rats given sheep anti-rat monocytes IgG (monocyte depleted), (D) rats injected with cobra venom factor (CVF) (complement depleted). Specificity controls for anti-cell antisera were performed in heterologous Masugi nephritis (PMN dependent) and accelerated Masugi nephritis (monocyte dependent). Complement and monocyte depletion significantly delayed the onset of proteinuria. PMN depletion had no effect. In the monocyte depleted group no differences in glomerular deposition of C3, C9 and MAC were seen in comparison to the nephritic control rats. The amount of anti-FX1A antibody bound was the same in all groups. This demonstrates that, besides complement, monocytes are necessary to induce renal damage in passive Heymann nephritis, a finding not previously reported. 1. SALANT, D. J., S. BELOK, M. P. MADAIO, and W. G. COUSER. 1980. A new role for

complement in experimental membranous nephropathy in rats. J. Clin. Invest. 66: 1339.

Institut fUr Immunbiologie, Arbeitsgruppen Elektronenmikroskopie und Zellulare Immunologie der Universitat Freiburg, D-7800 Freiburg, und Institut fur Organische Chemie der Universitat Tubingen, D-7400 Tubingen, FRG

G.24 Electron energy loss spectroscopy (EELS) as a novel method to localize the leukocyte activator Iipopeptide in the lymphoid cell line BCL 1 and in bone marrow-derived macrophages B. WOLF, S. HAUSCHILDT, B. UHL, J. METZGER, G. JUNG, and W. G. BESSLER Lipopeptide, a synthetic analogue of bacterial lipoprotein, is a potent activator for leukocytes and lymphoid cell lines. However, the fate of lipopeptide after the interaction with its

102 . XXth Meeting of the Society of Immunology target cells is as yet unknown. We applied the novel method of electron energy loss spectroscopy (EELS) to follow up the way of lipopeptides from the membrane to the interior of the cell, after interaction with the lymphoid cell line BeL 1 as well as in bone marrowderived macrophages. This method is based on the fact that electrons from the primary beam interact with the target atoms on passing the cell. The energy spectrum of the transmitted beam shows discontinuities (edges), of which the interpretation can give quantitative information about the elemental and molecular distribution of the sample within the cell. Our results show that lipopeptide is, at various times after stimulation, found distributed at different locations within the cell. The major amount is located within the membrane, further amounts are located in the cytoplasm, the nuclear membrane, and within the nucleus. Our results help to elucidate the molecular mechanism of lymphocyte stimulation by lipopeptide. In general, EELS constitutes a valuable method to localize lipopeptides as well as any given molecule (e.g. growth factors, drugs, pharmacal within cell compartments, tissues, and biological material.