Implication of gammadelta T cells in the human immune response to cytomegalovirus.

In normal individuals, gammadelta T cells account for less than 6% of total peripheral T lymphocytes and mainly express T-cell receptor (TCR) Vdelta2-Vgamma9 chains. We have previously observed a dramatic expansion of gammadelta T cells in the peripheral blood of renal allograft recipients only when they developed cytomegalovirus (CMV) infection. This increase was long lasting (more than 1 year), was associated with an activation of gammadelta T cells, and concerned only Vdelta1 or Vdelta3 T-cell subpopulations. Analysis of gammadelta TCR junctional diversity revealed that CMV infection in these patients was accompanied by (a) a marked restriction of CDR3 size distribution in Vdelta3 and, to a lesser extent, in Vdelta1 chains; and (b) a selective expansion of Vdelta1 cells bearing recurrent junctional amino acid motifs. These features are highly suggestive of an in vivo antigen-driven selection of gammadelta T-cell subsets during the course of CMV infection. Furthermore, Vdelta1 and Vdelta3 T cells from CMV-infected kidney recipients were able to proliferate in vitro in the presence of free CMV or CMV-infected fibroblast lysates but not uninfected or other herpes virus-infected fibroblast lysates. This in vitro expansion was inhibited by anti-gammadelta TCR mAb's. These findings suggest that a population of gammadelta T cells might play an important role in the immune response of immunosuppressed patients to CMV infection.

Different epitopes are required for gp130 activation by interleukin-6, oncostatin M and leukemia inhibitory factor.

Gp130 is the common signal transducing receptor subunit of interleukin (IL)-6, IL-11, leukemia inhibitory factor (LIF), oncostatin M (OSM), ciliary neurotrophic factor and cardiotrophin-1. IL-6 and IL-11 induce gp130 homodimerization whereas the others lead to the formation of heterodimers with LIFR or OSMR. Binding epitopes for IL-6 and IL-11 are located in the immunoglobulin-like domain and the cytokine binding module (CBM). Here we show that a gp130 mutant lacking domain 1, although unresponsive to IL-6 and IL-11, can still activate signal transducer and activator of transcription (STAT) transcription factors in response to LIF or OSM. Moreover, point mutations in the CBM of gp130 (F191E and V252D) that severely impair signal transduction in response to IL-6 and IL-11 differentially interfere with gp130 activation in response to LIF and OSM. Thus, epitopes involved in gp130 homodimerization are distinct from those leading to the formation of gp130/LIFR or gp130/OSMR heterodimers. These findings may serve as the base for rational design of gp130 antagonists that specifically interfere with bioactivity of distinct IL-6-type cytokines.

Production and characterization of monoclonal antibodies against the leukemia inhibitory factor low affinity receptor, gp190.

Leukemia inhibitory factor (LIF), oncostatin-M (OSM), ciliary neurotrophic factor (CNTF) and cardiotrophin-1 (CT1) act through transmembrane receptors which share the gp190 glycoprotein chain. The understanding of its involvement in the biology of these cytokines is of importance since these systems have recently been shown to participate in major inflammatory and neoplastic processes such as myelomatosis (Rose-John, S., Heinrich, P.C., 1994. Soluble receptors for cytokines and growth factors: generation and biological function. Biochem. J. 300, 281). In addition, this family of receptors also shares the gp130 transducing chain, with the IL6 and IL11 receptors. Because IL6 and gp130 were the first members to be discovered, most of the available reagents are directed at them. In this respect, monoclonal antibodies have played a major role in elucidating these receptor/ligand interactions and exploring the pathophysiological aspects of their biology. So far, no such reagents have been described for the gp190. We now report the production and characterization of 16 monoclonal antibodies directed against human gp190. They were obtained using recombinant chimeric or truncated proteins produced in a eukaryotic CHO cell line. One was able to block the biological activity of LIF. Because gp190 comprises two hematopoietin binding domains, crude epitope mapping was possible using the same reagents. While more of these antibodies are required, the present set validate the technological approach used for their preparation and should improve our understanding of this class of cytokines.

Leukemia inhibitory factor: part of a large ingathering family.

Leukemia Inhibitory Factor (LIF) has a wide variety of biological activities. It regulates the differentiation of embryonic stem cells, neural cells, osteoblasts, adipocytes, hepatocytes and kidney epithelial cells. It also triggers the proliferation of myoblasts, primordial germ cells and some endothelial cells. Many of these biological functions parallel those of interleukin-6, Oncostatin M, ciliary neurotrophic factor, interleukin-11 and cardiotrophin-1. These structurally related cytokines also share subunits of their receptors which could partially explain the redundancy in this system of soluble mediators. In vivo LIF proves important in regulating the inflammatory response by fine tuning of the delicate balance of at least four systems in the body, namely the immune, the hematopoietic, the nervous and the endocrine systems. Although we are far from its therapeutic applications, the fast increasing knowledge in this field may bring new insights for the understanding of the cytokine biology in general.

The presence in human serum of a circulating soluble leukemia inhibitory factor receptor (sgp190) and its evolution during pregnancy.

The specific binding of leukemia inhibitory factor (LIF) or oncostatin-M (OSM) to transmembrane gp190 or gp130 leads to their oligomerization which is necessary for signal transduction. Although, sgp190 and sgp130 counterparts also exist, their biological significance remains to be determined. Interestingly, sgp190 forms have been identified in large amounts in normal mouse sera but so far not in human sera. During gestation, murine sgp190 increases 20-fold to 30-fold, while sgp190 RNA levels increase mostly in the liver, uterus and placenta. Sandwich ELISAs were used to detect the presence and follow the evolution of LIF concentrations and its sgp190 receptor subunit in the sera of healthy pregnant and non-pregnant women. The LIF concentrations in whole blood of pregnant women were significantly lower than those of non-pregnant women, whereas they returned to these latter values soon after delivery. In 51 non-pregnant women, the mean sgp190 concentration was 4.3 0.3 ng/ml. It rose slowly but steadily, increasing 4-fold from the 4th to th 34th week of amenorrhea, at which time, it accelerated more rapidly until reaching 41 weeks of amenorrhea, 12 times the mean control value. Several hours prior to delivery, the sgp190 concentration dropped sharply before returning to control levels 2 days later. The marked increase of sgp190 measured in pregnant women extends to humans the observation previously made in mice and suggests a pivotal role for this cytokine system not only during implantation and early embryo development but also throughout pregnancy and more specifically in the biological function of placenta.

Stimulation of leukemia inhibitory factor receptor degradation by extracellular signal-regulated kinase.

Leukemia inhibitory factor (LIF) signals via the heterodimeric receptor complex comprising the LIF receptor alpha subunit (LIFRalpha) and the common signal transducing subunit for interleukin-6 cytokine receptors, gp130. This study demonstrates that in different cell types, the level of LIFRalpha decreases during treatment with LIF or the closely related cytokine oncostatin M (OSM). Moreover, insulin and epidermal growth factor induce a similar LIFRalpha down-regulation. The regulated loss of LIFRalpha is specific since neither gp130 nor OSM receptor beta shows a comparable change in turnover. LIFRalpha down-regulation correlates with reduced cell responsiveness to LIF. Using protein kinase inhibitors and point mutations in LIFRalpha, we demonstrate that LIFRalpha down-regulation depends on activation of extracellular signal-regulated kinase 1/2 and phosphorylation of the cytoplasmic domain of LIFRalpha at serine 185. This modification appears to promote the endosomal/lysosomal pathway of the LIFRalpha. These results suggest that extracellular signal-regulated kinase-activating factors like OSM and growth factors have the potential to lower specifically LIF responsiveness in vivo by regulating LIFRalpha half-life.

Identification of agonistic and antagonistic antibodies against gp190, the leukemia inhibitory factor receptor, reveals distinct roles for its two cytokine-binding domains.

The receptor for the cytokine leukemia inhibitory factor (LIF) associates the low affinity binding component gp190 and the high affinity converter gp130, both of which are members of the family of hematopoietic receptors characterized by the cytokine receptor homology (CRH) domain. The gp190 is among the very few members of this large family to contain two CRH domains. The membrane-distal one (herein called D1) is followed by an Ig-like domain, a membrane-proximal CRH domain called D2, and three type III fibronectin repeats. We raised a series of monoclonal antibodies specific for the human gp190. Among them was the blocking antibody 1C7, which was directed against the D1Ig region and which impaired the binding of LIF to gp190. Another blocking antibody, called 12D3, was directed against domain D2 and interfered with the reconstitution of the high affinity receptor complex, independently of the interaction between LIF and gp190. The blocking effect of these two antibodies concerned four cytokines known to use gp190, i.e. LIF, oncostatin M, ciliary neurotrophic factor, and cardiotrophin-1. Among 23 antibodies tested alone or in combination (two anti-D2 and 21 anti-D1Ig), only the mixture of the two anti-D2 antibodies displayed agonistic activity in the absence of the cytokine. Taken together, these results demonstrate that the two CRH domains of gp190 play different functions in ligand binding and receptor activation.

Interleukin-10 is a growth factor for human myeloma cells by induction of an oncostatin M autocrine loop.

We have a previously reported that interleukin-10 (IL-10) is a potent but IL-6-unrelated growth factor for freshly explanted myeloma cells (Lu et al, Blood 85:2521, 1995). We have also shown that exogenous IL-10 supported the growth of XG-1 and XG-2 human myeloma cell lines (HMCL) through an IL-6-independent mechanism. (Lu et al, Blood 85:2521, 1995). Because the IL-10 receptor does not involve the gp 130 IL-6 transducer, we have attempted to elucidate the mechanisms of IL-10 action on myeloma cells. Our results indicate that the myeloma cell growth factor activity of IL-10 was abrogated by an antibody to the gp 130 IL-6 transducer, indicating that it was mediated through one of the gp 130-activating cytokines. We found that myeloma cells from XG-1 and XG-2 HMCL and from 5 of 6 patients' tumoral samples produced oncostatin M (OM) constitutively but failed to produce IL-6, IL-11 and leukemia-inhibitory factor (LIF). The autocrine OM was inactive in the absence of IL-10 due to lack of a functional OM receptor on myeloma cells. IL-10, by inducing the receptor for LIF (LIFR), produced a functional autocrine OM loop in XG-1 and XG-2 cells and in primary myeloma cells from 2 patients. We also found that some myeloma cell lines (XG-4, XG-6, and XG-7) an fresh myeloma cells from 3 of 6 patients produced an autocrine IL-10 and that these cells constitutively expressed LIFR. One HMCL (XG-7) produced IL-10, OM, and IL-6 an expressed LIFR. The XG-7 cells used OM and IL-6 as autocrine growth factors. We have previously shown that IL-10 could induce IL-11 receptor in myeloma cells and confer on them sensitivity to IL-11 (Lu et al, FEBS Lett 377:515, 1995). Taken together, these results show that IL-10 is a key cytokine for inducing the expression of LIFR and IL-11R and possibly another uncharacterized OM coreceptor on myeloma cells and that OM and IL-10 might be produced by myeloma cells. They also emphasize that all myeloma cell growth factors reported to data involve an activation of the gp130 IL-6 transducer.

Epitope-function relationships of human leukemia inhibitory factor receptors using a novel set of anti-gp190 mAB.

The binding and functional properties of a set of six mAb directed against the human gp190 [leukemia inhibitory factor (LIF) receptor] signal transducing molecule were determined. Each of the antibodies reacted with a distinct epitope on gp190 expressed either by gp190-transfected Chinese hamster ovary cells or by the LIF receptor-positive choriocarcinoma JAR cell line. Two of the antibodies (1B4 and 6E6) had binding stoichiometries that were approximately 2-fold lower than those of other mAb (10B2, 12D9 and 7G7), suggesting either that gp190 is present as a pre-associated homodimer in the cell membrane or that part of gp190 is pre-associated with another component. Two mAb (1C7 and 1B4) were found to inhibit LIF binding on the two cell types studied. On JAR cells, this inhibition was, however, restricted to the high-affinity LIF component, suggesting different modes of LIF engagement with the low- and high-affinity receptor species. mAb 1C7 and 1B4 were also found to synergize for inhibiting LIF high-affinity binding. This synergy also extended to the inhibition of LIF- or oncostatin M (OSM)-induced proliferation of a Ba/F3 cell line co-transfected with human gp130 and gp190. However, this mAb combination inhibited LIF- but not OSM-induced haptoglobin secretion by HepG2 cells, suggesting that whereas haptoglobin secretion induced by LIF involves gp130/gp190 common LIF/OSM type I receptors, that induced by OSM mainly involves type II OSM receptors.

The mannose 6-phosphate/insulin-like growth factor II receptor is a nanomolar affinity receptor for glycosylated human leukemia inhibitory factor.

Comparison of the binding properties of non-glycosylated, glycosylated human leukemia inhibitory factor (LIF) and monoclonal antibodies (mAbs) directed at gp190/LIF-receptor beta subunit showed that most of the low affinity (nanomolar) receptors expressed by a variety of cell lines are not due to gp190. These receptors bind glycosylated LIF produced in Chinese hamster ovary cells (CHO LIF) (Kd = 6.9 nM) but not Escherichia coli-derived LIF or CHO LIF treated with endoglycosidase F. CHO LIF binding to these receptors is neither affected by anti-gp190 mAbs nor by anti-gp130 mAbs and is specifically inhibited by low concentrations of mannose 6-phosphate (Man-6-P) (IC50 = 40 microM), suggesting that they could be related to Man-6-P receptors. The identity of this LIF binding component with the Man-6-P/insulin-like growth factor-II receptor (Man-6-P/IGFII-R) was supported by several findings. (i) It has a molecular mass very similar to that of the Man-6-P/IGFII-R (270 kDa); (ii) the complex of LIF cross-linked to this receptor is immunoprecipitated by a polyclonal anti-Man-6-P/IGFII-R antibody; (iii) this antibody inhibits LIF and IGFII binding to the receptor with comparable efficiencies; (iv) soluble Man-6-P/IGFII-R purified from serum binds glycosylated LIF (Kd = 4.3 nM) but not E. coli LIF. The potential role of Man-6-P/IGFII-R in LIF processing and biological activity is discussed.

T-lymphocyte populations in hepatitis C and HIV co-infected patients treated with interferon-alfa-2a and ribavirin.

OBJECTIVE: The effects on T-lymphocyte populations of two interferon-alfa-2a (IFN) regimens associated with ribavirin were evaluated in 36 HCV-HIV co-infected patients with chronic hepatitis C, T-CD4 cell count > 250 cells/ micro L and a plasma viral load of < 10 000 HIV RNA copies/mL. METHODS: Patients were given IFN for 48 weeks. Group A (18 patients) received 6 mega units (MU) subcutaneously three times a week for 24 weeks, then 3 MU three times a week for the last 24 weeks. Group B (18 patients) received 9 MU daily for 2 weeks, 3 MU daily for 22 weeks, then 3 MU three times a week for the last 24 weeks. Serum HCV RNA was evaluated at weeks 12 and 72. Ribavirin was added at week 16 for virologic nonresponders at week 12. CD3, CD3 CD4, CD3 CD8, CD3 CD4 human leucocyte antigen (HLA)-DR and CD3 CD8 HLA-DR lymphocyte subsets were evaluated before, during and after treatment by cytofluorometry. Controls were healthy and HCV mono-infected patients. RESULTS: CD3 CD4 and CD3 CD8 T-cells counts were both impaired during anti-HCV therapy, but returned to baseline value after treatment completion. Lymphopenia concerned mainly CD8 T-cells, the percentage of which decreased, whereas that of CD4 increased. Three patients displayed reversible CD4 lymphopenia < 200 cells/ micro L. HIV infection at inclusion was responsible for higher CD3 CD8 HLA-DR T-cell percentages in co-infected patients than in healthy and HCV mono-infected subjects. T-cell sequestration in lymphoid tissues and enhanced apoptosis may account for lymphopenia. CONCLUSION: High-dosed IFN anti-HCV therapy induced only moderate and transient CD4 lymphopenia in HIV co-infected patients.

Identification of a Leu-lle internalization motif within the cytoplasmic domain of the leukaemia inhibitory factor receptor.

Leukaemia inhibitory factor (LIF) signals via a heterodimeric receptor complex comprised of the LIF receptor (LIFR) and the interleukin (IL)-6 signal transducer gp130. Upon binding to its cognate receptor LIF is internalized. In this study, we show that the LIFR is endocytosed independently of gp130. By using a heterochimaeric receptor system we identified a dileucine-based internalization motif within the cytoplasmic domain of the LIFR. Our findings suggest that a heterodimeric LIFR/gp130 complex and homodimeric gp130/gp130 complex are endocytosed via distinct internalization signals.

Binding of leukemia inhibitory factor (LIF) to mutants of its low affinity receptor, gp190, reveals a LIF binding site outside and interactions between the two cytokine binding domains.

The gp190 transmembrane protein, the low affinity receptor for the leukemia inhibitory factor (LIF), belongs to the hematopoietin family of receptors characterized by the cytokine binding domain (CBD). gp190 is one of the very few members of this family to contain two such domains. The membrane-proximal CBD (herein called D2) is separated from the membrane-distal one (called D1) by an immunoglobulin-like (Ig) domain and is followed by three fibronectin type III repeats. We used truncated gp190 mutants and a blocking anti-gp190 monoclonal antibody to study the role of these repeats in low affinity receptor function. Our results showed that the D1Ig region was involved in LIF binding, while D2 appeared to be crucial for the proper folding of D1, suggesting functionally important interactions between the two CBDs in the wild-type protein. In addition, a point mutation in the carboxyl terminus of the Ig region strongly impaired ligand binding. These findings suggest that at least two distinct sites, both located within the D1Ig region, are involved in LIF binding to gp190, and more generally, that ligand binding sites on these receptors may well be located outside the canonical CBDs.