THIRD INTERNATIONAL WORKSHOP ON CYTOKINES / 465

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ALLOANTIGEN PRESENTATION TO NAIVE CD8+ T CELLS BY CLONED POPULATIONS OF MOUSE SPLENIC MACROPHAGES: DIFFERENTIAL EXPRESSION OF COSTIMUIATOR MOLECULES DEFINES THE ALLOANTIGEN- PRESENTING PHENOTYPE. J.N. McCormack. D.&m and W.S. Walkec Department of ImmunoloRy!y St. Jude Children's Research Hospital, Memphis, Tennessee, USA 38101-0318.

About one-third of mouse SpLSlliC macrophage (M@) progenitors give rise to cloned progeny that consitutively induce the selective proliferation of naive allogeneic CD8+ T cells in a CD4+ helper cell-independent manner -- a response that is inhibited by mAb to the MHC class I molecules present on the Ma. Colony mixing experiments indicate that the failure of most Mm clones to present alloantigen is not due to their suppression of the ability of CD8+ cells to respond, nor do the nonpresenting clones interfere with the activity of the presenting Me. These t"o distinct presenting phenotypes were found to be a stable characteristic in a panel of cell lines derived from individual clones of MQ. Analysis of the cell lines reveals that the differential expression of presenting activity cannot be attributed to the levels of MHC class I molecules; rather. the cell lines and the primary Me clones differ in their expression of multiple costimulator molecules that likely function to induce, in part, the expression of IL-2 receptors on and the secretion of IL-2 by the T cells. Work is in progress to identify these molecules and determine the basis for their restricted expression. (Supported by AI-17979 from the NIH and by ALSAC).

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MECHANICAL SHEAR FORCES PRIME PBMC FOR INCREASED TNFa BUT NOT IL-l!3 SYNTHESIS. M.J. C.A. Dinarello. LIC. New England Medical Center and Massachusetts Institute of Technology, Boston MA 02111 USA.

Extracorporeal circulation exposes blood to mechanical shear forces. Numerous studies have reported effects of mechanical shear on blood cell morphology and function. Effects on cytokine production have not been addressed. In this study, we analyzed the effect of mechanical shear on the production of IL-lp and TNFa by human peripheral blood mononuclear cells (PBMC). Whole blood from 7 healthy donors was exposed to shear stress of 50 dyne/cm*, 200 dyne/cm*, or 500 dyne/cm2 for 5 min. PBMCs were separated from sheared blood samples and cultured for 24 hrs, then total IL-l!3 and TNFa measured by specific RIAs. LPS or S. epidermidis (S. epi) were added to some cultures to assess cell priming. Controls included unexposed and device-exposed, ““sheared blood. Cell viability was not affected by shear stress to 500 dyne/cm*. Cytokine production by PBMC exposed to shear alone was <loo-150 pgfml. IL-ID produced by LPS or S.epi-exposed cells was not altered by shear stress 2 200 dy”e/cm2. At 500 dyne/cm*. IL-lp levels were 70% of unexposed controls for LPS- exposed cells and 50% of unexposed controls for S.epi-exposed cells. In contrast, TNFa synthesis increased -2.fold Over

unexposed controls at 200 dyne/cm2 for cells exposed to either bacterial stimulant. At 500 dyne/cm*, TNFa production in LPS- exposed but not S. epi-exposed cells was also enhanced. Thus, exposure to mechanical force differentially affects IL-l!3 and TNFa synthesis by PBMC.

93

PREFERENTIAL STIMULATION OF IL-lp BY BORRELIA BURGDORFERI COMPARISON TO IL-la AND IL-lra. L.C.. E. Vannier.

C.A. Dm New England Medical Center, Tufts Univ, Boston MA 02111, USA

The Lyme disease spirochete, Borrelia burgdorferi (Bb), is a potent inducer of cytokines. particularly IL-lb. Because cytokines may contribute to the pathogenesis of Lyme disease, we investigated the induction and regulation of IL-lb and related cytokines by Bb in normal human peripheral blood mononuclear cells (PBMC). Cytokines were measured by specific RIAs. In response to live Bb (5 spirochetes/cell), PBMC of 23 normal donors synthesized 65fll “g/ml IL-lp, compared to 12f2 “g/ml induced by LPS [p<O.Ol] or 6fl “g/ml induced by killed Bb [P<O.Ol]. Live Bb induced >6 fold more IL-la than IL-la [p<O.OOl] and >7-fold more IL-lb than IL-lra [p<O.O06], while little difference was see” in the amount of these cytokines induced by LPS or killed Bb. I” contrast to LPS-induced IL-lb mRNA which peaked 3 hrs after stimulation, Bb-induced IL-l!3 mRNA showed a biphasic patter” with peaks at 3 hrs and 18 hrs. Bb-induced IL-lra mRNA was similar to that see” with LPS. By 4-6 hrs after stimulation. Bb induced IL-lb exceeded both Bb-induced IL-lra [p<.O4] and LPS-induced IL-ID [p<O.O2] by 6- fold. The kinetics and amounts of IL-la and IL-lra induced by the 2 stimulants did not differ. In dose-response experiments, synthesis of IL-lra did not parallel the increased synthesis of IL-la in response to increasing numbers of spirochetes. This preferential synthesis of IL-lp compared to IL-lra induced by Bb is the first example of excess agonist over antagonist production by a microbial pathogen.

94

IL-lb SYNTHESIS BY PBMC DIFFERS IN RESF’ONSE TO INFECTIVE AND NON-INFECTIVE STRAINS OF BORRELIA BURGDORFERI. kC.

s. S. Isa. A.C. Steere. M.S. w. C.A. Dtn& New England Medical Center. Tufts Univ, Boston MA 02111 USA.

The Lyme disease spirochete Borrelia burgdorferi (Bb) is a potent inducer of IL-lb, a cytokine which may contribute to disease pathogenesis. Because Bb strains lose infectivity [J Inf Dis,163,‘91] after 15-20 laboratory passages, we studied whether differences in infectivity related to cytokine production. The production of cytokines by PBMC of 14 normal donors in response to 3 different strains of Bb was studied. Strains included 297 (a spinal fluid isolate), N40 (a tick isolate), and G39/40 (a laboratory adapted strain). PBMC from 6 additional donors were tested with cloned Bb derived from the same strains, including both low passage [LPI and high passage [HP] 297. Bb were incubated (5 spirochetes/PBMC) for 24 hr with PBMC, then total synthesis of IL-ID. IL-la, TNF, IL-6, and IL-lra was measured by specific RIAs. In viva infectivity of strains was determined after inoculation into mice by recovery of live Bb after 3 wks. Results: Bb strain 297 induced significantly more IL-lp than N40 or G39/40 (llOf19 “g/ml compared to 55f13 “g/ml [p<O.Ol] and 36f12 “g/ml [p<O.Ol]). Cloned LP 297 induced 82f6 “g/ml IL-lp; the 3 other clones all induced 30-32 “g/ml IL-I!3 (p<O.O05). No differences were see” in synthesis of IL-la, TNF, IL-6, or IL-lra by the strains or clones. LP clones were infective, while HP clones were not infective. Differences in IL-lp-inducing capacity of Bb strains may account for some of the variable clinical manifes-tations of Lyme disease, and may relate to infectivity of the organism in vim.

95

lit-i0 INTERNATIclNAL COLLABOFATIvE STUDIES FOR THE STANDARDISATION OF CYTOICINES: THRIR ROLE IN THE RESEARCH AND CLINICAL "SE OF CYTOKINES. A R Mire-Sluis and R Thorpe. Division of Immunobiology, NIB.%, Blanche Lane, South Nimms, Herts., EN3 6QG, UK. During recent years, the growing awareness of the role of cytokines in the pathogenesis and treatment of disease has stimulated an already rapidly growing research interest in these factors. The advent of rapid cloning and screening technology has meant that new cytokines are being isolated, cloned and produced in large quantities in very short time periods. A situation has now arisen whereby many different recombinant and natural forms of cytokines are available for research and clinical "se. If valid comparisons are to be made between the biological properties of these different forms, This

it is vital to assign a defined biological potency. is particvlarly important when comparing research

studies and when these cytokines are being used by clinicians where biological potency between different products must be correctly established. The WHO International Collaborative studies aim to assign a single worldwide unitage to each individual cytokine based upon their biological potency and immunological reactivity. The collaborative studies are also a unique opportunity to investigate how these multiple forms Of a cytokine vary in biological activity as well as assessing the accuracy, sensitivity and specificity of valuable assay systems. Therefore international standards Dlav an inumrtant role in unifying cytokine research woridwide anb we encourage all laboratories that possess cytokine responsive systems to take part in future collaborative studies.

96

ENHANCED PRODUCTION OF TUMOR NECROSIS FACTOR (TNF) BY HUHAN MONOCYTES EXPOSED TO HUMAN

DEFENSIN HNP-1. N.Mi5uno.T. Eolesnik.ova, 1. Gd”Z. R. Lehrer, N. Voitenok. Byelorussia" Inst. Blood Transfusion, Minsk. School of Medicine. University of California. Los Angeles. Inst. Experimental Hematology & Riotechnologv, Moscow *

Defensins.smal! catlonic peptidea with broad antibacterial specr-a and possible immunoregulatory function, are abundant in the cytoplasmic granules of neutroohils.. We studied the influence of human defensin HNP-1 on the synthesis of tumnr necf-osis factor (TNF) by human blood monocytes activated by Staphvlococcua aureu5 Cowan 1 (SAC). It wa-5 shown that TNF production wa6 augmented in pre4ence Of HNP-1 at concentration of

t$= 10 -

10 . HNF-1 alone induced no synthesis of TNF-a . It was sQgwn that high concentrations of HNF-1 (above 10 II) were cytotoxic far human monocytes. Our finding suggests that human neutrophil granule defensin HNP-1 can positively af feet TNF-a production by activated human monocy te5.