THIRD INTERNATIONAL WORKSHOP ON CYTOKINES / 469

115 EFFECT OF THERMAL INJURY ON TUMOR NECROSIS FACTOR-ALPHA PRODUCTION BY PERITONEAL MACROPHAGES IN A MURINE MODEL. u Somers and A& Biornson. James N. Gamble Institute of Medical Research, Cincinnati, Ohio 45219.

Little information exists regarding tumor necrosis factor- alpha (TNFa) production following severe trauma in the absence of bacteremia. To address this issue. we used a model of full- thickness scald injury (22% total surface area burn) in C57BL/6NCR mice; blood cultures were negative and mortality rates were low (<5%) over 7 days postburn. We examined TNFa production by peritoneal macrophages (MB) induced by i.p. injection of sterile thioglycollate broth 2 days prior to injury, with MB harvested 24 hours later. MB from either injured or sham-treated mice did not spontaneously produce TNFa. MB from both treatments produced TNFa at similar rates for 5 hours upon exposure to 100 rig/ml lipopolysaccharide (LPS). After 24 hours with LPS, TNFa activity was significant- ly reduced in cultures of Me from injured mice (<20% of maxi- mum), while activity produced by Ma from sham-treated mice remained elevated. Loss of soluble bioactivity was not parall- eled by increased Ma cell-associated TNFa activity. Similar observations were made when 300 #g/ml zymosan was used as a stimulus. Western blots of 24 hour culture media demonstrated that cultures of MB from injured mice contained less TNFa. These results suggest that stimulated Ms can produce TNFm after thermal injury, but mechanisms exist to down-modulate expression of bioactivity. This mechanism may protect the host from the deleterious effects associated with TNFa.

116

IL-4 PRIMES HUMAN MONONUCLEAR CELLS TO ENHANCED RESPIRATORY BURST - SYNERGISM WITH 1FN-a. MSommerlund, SEllermann-Eriksen. S.Haahr. S.C.Mooensen. GMoller-Larsen, Imstiture of Medical Microbiology, University of Aarhus, DK-8000 Aarhus C.

The effect of IL4 and other cytokines on the priming of mononuclear cells for respiratory burst has been elucidated. Cultures of freshly isolated human mononuclear cells were either incubated with recombinant IL4 alone or in combination with other cytokines: IFN-a, ILl, IL2, IL6, GM-CSF, TNF-a for 4 to 48 hours after which the respiratory burst was measured by luminol dependent chemiluminescence after triggering with PMA. During in vitro differentiation of mononuclear phagocytes, their capacity for respiratory burst declined. This decline was especially pronounced during the first 48 hours. We demonstrated that IL4 or IFN-a alone in a dose-dependent way primed mononuclear cells for an enhanced capacity for respiratory burst after 13 to 24 hours of in- cubation by slowing down the spontaneous decline of the response. When the cells were challenged with both IL-4 and IFN-a, a synergistic effect on priming was seen. This effect was not seen when IL-4 was combined with IL-l, IL-2, IL-6, GM-CSF or TNF-a. Antibody against IFN-gamma reduced the respiratory burst capacity of IL-4 primed cells in a dose-dependent manner. Thus the stimulating effect of IL4 seemed to be dependent of IFN-gamma. In conclusion our results suggest that a local secretion of IL4 in a region of inflammation may delay the decline of oxidative burst capacity of newly recruited mononuclear cells.

117

THE REQUIREMENT FOR A FETAL BOVINE SERUM COMPONENT AND THE INHIBITORY ROLE OF INTERLEUKIN-6 ON MACROPHAGE PROLIFERATION. C.C. Et&y Roswell Park Cancer Institute. Carlton and Elm Streets Buffalo, NY 14263

We have shown that there are two forms of progenitor cells for macrophages. The first is characterized by a short lag period (about one day) before initiating cell cycle and appears to be in the nonadherent fraction. These progenitor cells, found primarily in the bone marrow, form large colonies in liquid culture medium containina 20% fetal calf serum and 20% L-929 cell conditioned medium when cultured for seven days. The second progenitor cell, found primarllv in the adherent cell fraction of bone marrow and in peripheral tissues, form small colonies in 14 days. Proliferation of both progenitor cells requires CSF-1 and a component found in fetal calf serum but not in calf serum. We investigated the possibility that interleukin-6 was this component and that it might act as a competence factor as suaaested by others. To our surprise we found that interleukin-6 (lL6j inhibited the proliferation of both types of progenitor cells. This inhibitory effect was reversible since macrophages could initiate a proliferative response after the removal of IL6.from the culture medium. The introduction of anti-116 to IIISCrODhaOS CultureS Containina IL6 allowed for oroliferation indicatktg the effect was IL6 specifb. These results suggest that IL6 may play a regulatory role in viva by suppressing the production of bone marrow and tissue macrophages. Supported by grant Al 19490

118

THE UNIQUE REPERTOIRE OF CELL SURFACE PROTEINS ON MYELOID UNEAGE CELLS. C.C.Stewarl and SJ. Stewart Roswell Park Cancer Institute, Carlton and Elm Streets, Buffalo, NY 14263

The myeloid system is composed of the granulocytic and macrophage lineages. These functional end cells are produced from the proliferation and differentiation of a common myeloid progenitor cell. This cell differentiates into neutrophils, eosinophils. basophils, mast cells, and macrophages. As these cells differentiate they lose surface proteins characteristic of the common progenitor and acquire characteristics that are unique to the lineage. As the cells differentiate they also acquire the ablllty to respond to their lineage specific growth factors. For myelold ceils the factors include CSF?. GM-CSF. G-CSF. IW. If5 and some that have vet to be characterized more completely. The repertoire of surface molecules for each lineage and for each differentiation stage within a lineage can be determined simultaneously in a single panel using the appropriate antibodies to these cell surface proteins. We found that a panel of 4 antibodies could be used simultaneously to define these myeloid cell subsets. Macrophages and eoslnophils both express F4/6g but eosinophils can be distinguished from macrophages because they, like neutrophlls also express HK1.4. Neutrophils can be distinguished from eosinophils because they do not express F4/80. Mast cells and basophils express 854.2 and this molecule is not SXDreSSed bv anv of the other mvelold cells. The immature progenitor ceils can be recognized because they all express thy 1.2. Thus, only 4 antibodies-when used in combination -can simultaneously resolve all myeloid lineage cells and their precursors. Supported by Grant #Al 19490 from NIH

119

IN VITRO PRObUCTlON OF TNFa BY MURINE MACROPHAGES (MTf) STIMULATED By ATPEKtZL& fUMMT& CONIDIA. D. Taramelli. M.G. Melabarba. C. Sala. L. Riviera. M. Ghione and G. Cocuzza. lstituto di Mfcrobiologia. Universita’ di Milsno. Milsno. Italy I-20133

4. r&r@@ f’,+r./.‘ts 3 pathogenic fungus responsible for the dtse%ses allergic pulwnary aspergillosis end aspergilloma, as well 3s invasive dspergillosis particularly in tmmurocompromtsed hosts, both tn humans and rodents. In mrm3l subj.&s, the resolution of AI infections seems to be mediated by MO and polymurphonuclcsr cells (PMN). However, little is known about the role played by rrttlammatory medistors,suchas lymphokines in this process To address this issue, we evalvsted -vitrohe production of TNFo by mouse MU stimulated with wnidia of d fin 3 72 hr, MTT cytotoxicity assay against WEHI 164. Clorr3 13 cell lifts. We found that A. Ccontdta either alive or heat-killed were able to induce the release of TNFe in 3 boss-dependent manner by both restdent and peptone-induced MO from CD1 mtcs. Maximum levels, ranging between 800-500 U/ml of TNFa were obtained It MO. coaidia rstio of 16 and1 :4. TNFoc was detectable after 2 hr of stimulation and Its production increased over 3 24-48 hr period. The supernntnnts from A. r! comdia or Mu cultured in rn3dium only did not show any cytotoxic activity in the TNF bio3s3ay. We postulate that TNFor in normal subjects may contribute to the elimination of A. 7 conidia by increasing the activation of MO and by recruiting PMN at the site of tnfectien. These findinqs may help to set up immunotherapy protocols to potentiste the natural defenses of immunedepressed patients against rnfections by opportuntsttc fungi.

120

EXPRESSION OF MEMBRANE-BOUND CSF-1 BY T CELLS: POSSIBLE INVOLVEMENT IN THEIR INTERACTION WtTH ANTIGEN-PRESENTING CELLS. B. Tart-. E. Ben-Yair and E. Zisman. Department of Chemical Immunology, The Weizmann Institute of Science, Rehovot 76100, Israel

Colony stimulating factor-l (CSF-I) is a disulphide-linked homodimeric glycoprotein that is required for the growth and differentiation of mononuclear phagocytes, and is also involved in modulating various activities in mature cells. Interested by the possible immunoregulatory function of CSF-1, we assessed its effect in an assay of antigen presentation to T cell lines. Addition of CSF-1 lo the T cell proliferation assay at the onset of the cultures, inhibited the antigen-specific proliferative responses of two different T cell lines. The inhibition was dependent on CSF-1 concentrations and on the time of its addition to the cultures. Moreover, anti-CSF-1 antibodies seemed to exert a similar blocking activity. In addition, CSF-1 activity was detected in the T cell lysates and was found not to be secreted to the culture medium. Attempting the reconciliation of these sets of evidence, led us to reveal the expression of CSF-1 protein on the outer membranes of these two T cell lines. We thus propose that T cell CSF- 1 may participate in/or contribute to the interaction of T cells with CSF-1 receptor-bearing antigen-presenting cells.