Didier Heumann

Expression of MHC class II molecules contributes to lipopolysaccharide responsiveness

Activation of phagocytes by lipopolysaccharide (LPS) causes synthesis and secretion of various mediators of inflammation. CD14, a glycosylphosphatidylinositol‐anchored monocytic antigen serving as receptor for LPS, and members of the family of Toll‐like receptors mediate cellular activation in response to LPS. Here we investigated whether expression of MHC class II molecules modified the response to LPS. Comparing LPS responsiveness of human and murine cells differing for expression of MHC class II molecules, we found that lack or a low level of expression of MHC class II molecules resulted in diminished secretion of pro‐inflammatory cytokines following stimulation with LPS. Thus, expression of MHC class II molecules modifies LPS responsiveness, a finding suggesting that these molecules contribute to the pathogenesis not only of exotoxin‐triggered toxic shock but also of endotoxin‐triggered septic shock. Additionally to their role in antigen‐specific immunity MHC class II molecules may influence the inflammatory response triggered by microbial constituents.

Monoclonal antibody against a “Pan-T-Cell” antigen expressed by thymocytes, peripheral T-lymphocytes and T-cell leukemias

A monoclonal antibody, LAU-A1, which selectively reacts with all cells of the T-lineage, was derived from a fusion between spleen cells of a mouse immunized with paediatric thymocytes and mouse myeloma P X 63/Ag8 cells. As shown by an antibody-binding radioimmunoassay and analysis by flow microfluorometry of cells labelled by indirect immunofluorescence, the LAU-A1 antibody reacted with all six T-cell lines but not with any of the B-cell lines or myeloid cell lines tested from a panel of 17 human hematopoietic cell lines. The LAU-A1 antibody was also shown to react with the majority of thymocytes and E-rosette-enriched peripheral blood lymphocytes. Among the malignant cell populations tested, the blasts from all 20 patients with acute T-cell lymphoblastic leukemia (T-ALL) were found to react with the LAU-A1 antibody, whereas blasts from 85 patients with common ALL and 63 patients with acute myeloid leukemias were entirely negative. Examination of frozen tissue sections from fetal and adult thymuses stained by an indirect immunoperoxidase method revealed that cells expressing the LAU-A1 antigen were localized in both the cortex and the medulla. From the very broad reactivity spectrum of LAU-A1 antibody, we conclude that this antibody is directed against a T-cell antigen expressed throughout the T-cell differentiation lineage. SDS-PAGE analysis of immunoprecipitates formed by LAU-A1 antibody with detergent lysates of radiolabeled T-cells showed that the LAU-A1 antigen had an apparent mol. wt of 76,000 under non-reducing conditions. Under reducing conditions a single band with an apparent mol. wt of 40,000 was observed. Two-dimensional SDS-PAGE analysis confirmed that the 76,000 mol. wt component consisted of an S-S-linked dimeric complex. The surface membrane expression of LAU-A1 antigen on HSB-2 T-cells was modulated when these cells were cultured in the presence of LAU-A1 antibody. Re-expression of LAU-A1 antigen occurred within 24 hr after transfer of the modulated cells into antibody-free medium.

Production of tumor necrosis factor-α by naive or memory T lymphocytes activated via CD28

Abstract While it is well established that activated T cells can produce tumor necrosis factor alpha (TNF-α), it is less clear whether this function is confined to a given subset, e.g., memory cells. To approach this question, we investigated the production of TNF-α by human peripheral blood T lymphocytes activated with anti-CD28 mAb since this activation pathway is known to potentiate cytokine production. Under the culture conditions used, the amount of TNF-α produced was markedly enhanced compared to that obtained after activation with immobilized anti-CD3 mAb. The enhancement of TNF-α production was already apparent after incubation of T cells for 6 hr. Up to 5 ng/ml of TNF-α was measured on Day 2 in supernatants of cultures of 104 T lymphocytes. To determine the source of the cells producing high amounts of TNF-α, T lymphocytes were separated into two subpopulations, namely naive cells (expressing the CD45RA isoform) and memory cells (expressing the CD45RO isoform). While both subpopulations proliferated equally well after stimulation with anti-CD28 mAb, up to 90% of the TNF-α produced under these conditions originated from memory T cells. These results thus document that T cell activation via CD28 results in a marked increase in TNF-α production without affecting the functional disparity that exists between naive and memory T cells.

Molecular interactions of Leishmania promastigote surface protease with human α2-macroglobulin

The interaction of Leishmania promastigote surface protease (PSP) with the plasmatic protease inhibitor α2-macroglobulin (α2M) was investigated. In plasma, solubilized PSP forms covalent complexes only with α2M, at the exclusion of other protease inhibitors. The formation of complexes is accompanied by the proteolytic cleavage of the α2M subunit and by the transition from the ‘slow’ to the ‘fast’ form of α2M. The proteolytic activity of solubilized PSP on azocasein is inhibited by α2M. In contrast, we found no evidence for a specific interaction of α2M with the surface of promastigotes and PSP proteolytic activity on intact cells was not inhibited by α2M.

Human α2-macroglobulin as an inhibitor of insoluble trypsin

Abstract α2-Macroglobulin binds to insoluble trypsin bound on agarose beads inducing a reduction of proteolytic activity of the enzyme towards large substrates such as azocasein. When trypsin was bound on other matrices like sheep red blood cells or latex beads, the inhibition of proteolytic activity by α2-macroglobulin was complete. These results show that α2-macroglobulin inhibits similarly both soluble and insoluble proteinases.

Analysis of cytokine immune response profile in response to inflammatory stimuli in mice with genetic defects in fetal and adult hemoglobin chain expression

Injections of a crude fetal sheep liver extract (FSLE) containing fetal hemoglobin, MPLA, and glutathione (GSSH) reversed cytokine changes in aged mice. To investigate the role of fetal hemoglobin we derived mice with homzygous deletions for either of the two major βchains, HgbβmaKO or HgbβmiKO. Hgbβmi is the most prominent fetal Hgbβ chain, with Hgbβma more prominent in adult mice. Mice lacking another fetal Hgb chain, HgbεKO, died in utero. CHO cells transfected with cloned Hgb chains were used to produce proteins for preparation of rabbit heteroantibodes. Splenocytes from HgbβmaKO mice stimulated in vitro with Conconavalin A showed a higher IL-2:IL-4 ratio than cells from HgbβmiKO mice. Following immunization in vivo with ovalbumin in alum, HgbβmaKO mice produced less IgE than HgbβmiKO mice, suggesting that in the absence of HgbβmiKO mice had a predeliction to heightened allergic-type responses. Using CHO cells transfected with cloned Hgb chains, we found that only the fetal Hgb chain, Hgbε, was secreted at high levels. Secretion of Hgbβma or Hgbβmi chains was seen only after genetic mutation to introduce the two N-linked glycosylation sites present in Hgbε, but absent in the Hgbβ chains. We speculated that a previously unanticipated biological function of a naturally secreted fetal Hgb chain may be partly responsible for the effects reported following injection of animals with fetal, not adult, Hgb. Mice receiving injections of rabbit anti-Hgbε but not either anti-Hgbβma or anti-Hgbβmi from day 14 gestation also showed a bias towards the higher IL-2:IL-4 ratios seen in HgbβmiKO mice.

Regulation of fetal hemoglobin expression during hematopoietic stem cell development and its importance in bone metabolism and osteoporosis

&NA; We have shown that an altered tissue redox environment in mice lacking either murine beta Hemoglobin major (Hgb&bgr;maKO) or minor (Hgb&bgr;miKO) regulates inflammation. The REDOX environment in marrow stem cell niches also control differentiation pathways. We investigated osteoclastogenesis (OC)/osteoblastogenesis (OB), in bone cultures derived from untreated or FSLE‐treated WT, Hgb&bgr;maKO or Hgb&bgr;miKO mice. Marrow mesenchymal cells from 10d pre‐cultures were incubated on an osteogenic matrix for 21d prior to analysis of inflammatory cytokine release into culture supernatants, and relative OC:OB using (TRAP:BSP, RANKL:OPG) mRNA expression ratios and TRAP or Von Kossa staining. Cells from WT and Hgb&bgr;maKO mice show decreased IL‐1&bgr;,TNF&agr; and IL‐6 production and enhanced osteoblastogenesis with altered mRNA expression ratios and increased bone nodules (Von Kossa staining) in vitro after in vivo stimulation of mRNA expression of fetal Hgb genes (Hgb&egr; and Hgb&bgr;mi) by a fetal liver extract (FSLE). Marrow from Hgb&bgr;miKO showed enhanced cytokine release and preferential enhanced osteoclastogenesis relative to similar cells from WT or Hgb&bgr;maKO mice, with no increased osteoblastogenesis after mouse treatment with FSLE. Pre‐treatment of WT or Hgb&bgr;maKO, but not Hgb&bgr;miKO mice, with other molecules (rapamycin; hydroxyurea) which increase expression of fetal Hgb genes also augmented osteoblastogenesis and decreased cytokine production in cells differentiating in vitro. Infusion of rabbit anti‐ Hgb&egr; or anti‐ Hgb&bgr;mi, but not anti‐Hgb&agr; or anti‐ Hgb&bgr;ma into WT mice from day 13 gestation for 3 weeks led to attenuated osteoblastogenesis in cultured cells. We conclude that increased fetal hemoglobin expression, or use of agents which improve fetal hemoglobin expression, increases osteoblast bone differentiation in association with decreased inflammatory cytokine release. HighlightsOsteoclastogenesis is attenuated by increasing expression of fetal hemoglobins.Increased osteoblastogenesis is associated with decreased inflammatory cytokine release.Deletion of adult hemoglobin beta chain gene, Hgb&bgr;maKO, increased osteoblastogenesis.Injection of rabbit anti‐ Hgb&egr; or anti‐ Hgb&bgr;mi attenuated osteoblastogenesis.