Stromal-derived factor-1 in human tumors recruits and alters the function of plasmacytoid precursor dendritic cells.

Dendritic-cell (DC) trafficking and function in tumors is poorly characterized, with studies confined to myeloid DCs (DC1s). Tumors inhibit DC1 migration and function, likely hindering specific immunity. The role of plasmacytoid DCs (DC2s) in tumor immunity is unknown. We show here that malignant human ovarian epithelial tumor cells express very high levels of stromal-derived factor-1, which induces DC2 precursor (preDC2) chemotaxis and adhesion/transmigration, upregulates preDC2 very late antigen (VLA)-5, and protects preDC2s from tumor macrophage interleukin-10-induced apoptosis, all through CXC chemokine receptor-4. The VLA-5 ligand vascular-cell adhesion molecule-1 mediated preDC2 adhesion/transmigration. Tumor preDC2s induced significant T-cell interleukin-10 unrelated to preDC2 differentiation or activation state, and this contributed to poor T-cell activation. Myeloid precursor DCs (preDC1s) were not detected. Tumors may weaken immunity by attracting preDC2s and protecting them from the harsh microenvironment, and by altering preDC1 distribution.

Interleukin 4 counteracts the interleukin 2-induced proliferation of monoclonal B cells.

B cells from patients suffering from B-type chronic lymphocytic leukemia (B-CLL) are susceptible to the effects of several interleukins. Using the cells from 12 different patients we show that IL-4 does not synergize with anti-mu antibody for the enhancement of DNA synthesis. Moreover IL-4 profoundly (90%) suppresses the response to IL-2 in the 10 patient responders to this interleukin. This suppression occurs whether IL-2 is used alone, in costimulation with anti-mu antibody, or in synergy with IFN-gamma. In no instance did IL-4 induce terminal differentiation. This negative effect of IL-4 can take place in monoclonal B-CLL cells where IL-4 enhances the expression of CD23. IL-4 does not interfere with the upregulation of CD25 by IL-2. Thus, IL-4 may display inhibitory effects on the proliferative response of selected B cell populations. The antagonism between IL-4 and IL-2 has important implications for the potential use of cytokines in the management of B-CLL patients.

Role of interleukin 10 in the B lymphocyte hyperactivity and autoantibody production of human systemic lupus erythematosus.

Interleukin-10 (IL-10) is produced at a high level by B lymphocytes and monocytes of patients with systemic lupus erythematosus (SLE). In the present work, we analyzed whether this increased production of IL-10 contributed to the abnormal production of immunoglobulins (Ig) and of autoantibodies in SLE. The role of IL-10 was compared with that of IL-6, another cytokine suspected to play a role in these abnormalities. The spontaneous in vitro production of IgM, IgG, and IgA by peripheral blood mononuclear cells from SLE patients was weakly increased by recombinant IL (rIL)-6, but strongly by rIL-10. This production was not significantly affected by an anti-IL-6 mAb but was decreased by an anti-IL-10 mAb. We then tested the in vivo effect of these antibodies in severe combined immunodeficiency mice injected with PBMC from SLE patients. The anti-IL-6 mAb did not significantly affect the serum concentration of total human IgG and of anti-double-stranded DNA IgG in the mice. In contrast, the anti-IL-10 mAb strongly inhibited the production of autoantibodies, and, to a lesser extent, that of total human IgG. These results indicate that the Ig production by SLE B lymphocytes is largely IL-10 dependent, and that the increased production of IL-10 by SLE B lymphocytes and monocytes may represent a critical mechanism in the emergence of the autoimmune manifestations of the disease.

Depressed primary in vitro antibody response in untreated systemic lupus erythematosus. T helper cell defect and lack of defective suppressor cell function.

The in vitro antibody response of peripheral blood lymphocytes (PBL) from 19 patients with untreated systemic lupus erythematosus (SLE) was compared with that of 20 control patients and 44 normal subjects. Trinitrophenyl polyacrylamide beads (TNP-PAA) were used to induce IgM anti-TNP plaque-forming cells. SLE patients displayed a markedly depressed, and in most instances virtually absent, response. This was not due to an unusual kinetics of the response; nor could it be induced by preincubation of SLE patients' PBL. In co-cultures of SLE patients and normal PBL, the former, with few exceptions, did not exert a suppressive effect. In four patients the anti-TNP response of either unfractionated or T-depleted SLE PBL could be restored by T cells from a normal individual. Conversely in three of these patients, SLE T cells could not support the response of normal B cells, suggesting a T helper cell defect in SLE PBL. Concanavalin A (Con A)-induced suppressor cells of the antibody response could be assayed by two approaches: (a) in responder SLE patients, by the direct addition of Con A to TNP-PAA-stimulated cultures; (b) in seven patients by transfer of Con A-activated cells to the responding culture of a normal allogeneic donor. In both cases SLE PBL were able to exert a suppressive effect to the same extent as normal PBL.

Production of interleukins in human immunodeficiency virus-1-replicating lymph nodes.

To document the in vivo interactions occurring between the immune system and HIV replicating cells, we analyzed using in situ hybridization the production of IL-1 beta, IL-6, IL-2, and INF-gamma in eight hyperplastic lymph nodes from HIV-1 infected patients. Numerous IL-1 beta- and IL-6-producing cells associated in clusters were detected in sinuses. Few individual IL-1 beta- and IL-6-producing cells were present in interfollicular and follicular areas. IL-2- and INF-gamma-producing cells were observed in all lymph node compartments, with a selective enrichment in germinal centers. The amount and distribution of IL-1 beta, IL-6-, and IL-2-producing cells in HIV lymph nodes were not different from those found in six HIV unrelated hyperplastic lymph nodes. In contrast, a higher level of INF-gamma production was observed in HIV-1 lymph nodes. The CD8+ cells that accumulate in germinal centers of HIV lymph nodes (and not in non-HIV germinal centers) were actively involved in this INF-gamma production. INF-gamma synthesizing cells were in direct contact with cells containing HIV core antigens and HIV RNA. Thus a high INF-gamma production may characterize anti-HIV T cell immune response, potentially contributing to control of viral spreading as well as to the development of follicle lysis.

High Mda-7 expression promotes malignant cell survival and p38 MAP kinase activation in chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL)-B-cells are quiescent differentiated cells that produce interleukin (IL)-10 and accumulate due to resistance to apoptosis. The mechanisms underlying such resistance are poorly understood. Herein we show that all CLL B-cells tested (30/30) display high mRNA and protein expression of the tumor suppressor Mda-7/IL-24, an IL-10 family member, in comparison to normal B cells. A downstream Mda-7 signaling target, p38 mitogen-activated protein kinase (MAPK) was highly phosphorylated in all CLL cells but not in normal B-cells. Mda-7 expression and p38 MAPK phosphorylation diminished in culture and the latter could be reinduced by recombinant (r)-IL-24 or LPS and Mda-7 transfection. Mda-7/IL-24 siRNA specifically inhibited p38 MAPK phosphorylation in CLL without affecting p38 MAPK, bcl2, or Lyn expression, further demonstrating the direct role of Mda-7/IL-24 in p38 MAPK activation. Both pharmacological inhibition of p38 MAPK and Mda-7 silencing augmented spontaneous apoptosis by three-fold in CLL cells cultured in autologous serum, which was reversed by LPS and r-IL-24. We established the role of p38 MAPK in CLL cell survival and demonstrated a paradoxical effect, whereby Mda-7 and IL-24, inducers of apoptosis in diverse cancer cells, promote the survival of CLL B-cells through p38 MAPK activation.

Intrapleural production of interleukin 6 during mesothelioma and its modulation by gamma-interferon treatment.

In vivo production of monokines was analyzed in 17 human malignant pleural mesotheliomas. High concentrations of interleukin 6 (IL-6) were detected in pleural effusions, contrasting with low levels of IL-1 beta and tumor necrosis factor alpha. This production arose from malignant cells, as shown by immunochemical analysis of pleural cells and by production of IL-6 by mesothelial cell lines. Intrapleural administration of recombinant human gamma-interferon to six patients led to a marked decrease in intrapleural IL-6 concentrations in all cases. This treatment was associated with in situ activation of macrophages and cytotoxic T-lymphocytes, as indicated by increased intrapleural neopterin and soluble CD8 concentrations. In vitro gamma-interferon had no effect on the production of IL-6 by mesothelial cell lines but decreased the growth of 3 of 6 mesothelioma cell lines. These results indicate that systemic manifestations of malignant mesothelioma, including fever, cachexia, and thrombocytosis may be related to the production of IL-6 by malignant cells, and that local gamma-interferon infusion may reduce this production by stimulating antitumoral immunity and/or by directly decreasing the proliferation of malignant cells.

JAK3 associates with the human interleukin 4 receptor and is tyrosine phosphorylated following receptor triggering.

In human B cells, interleukin 4 (IL4) acts in regulating proliferation, antigen expression, isotype switching and differentiation. These different effects are mediated through the IL4R complex including the IL2R gamma chain (gamma c) and a specific p130/140 binding unit referred below as human Interleukin 4 Receptor (IL4-R). Here, we studied the signal transduction events following IL4R activation and leading to CD23 expression on resting B cells. We demonstrate that IL4R triggering induced the tyrosine phosphorylation of JAK3 and of a p170 protein. Coimmunoprecipitation of JAK3 with the IL4R suggests a physical association which exists prior to IL4R complex stimulation. Orthovanadate treatment, while having no effect on IL4-induced p130 phosphorylation, leads to the hyperphosphorylation of the p170 and inhibits IL4-induced CD23 expression. These suggest that two mandatory steps exist in early IL4 signaling: one controlled by JAK3 activation and the other by the p170 phosphoprotein.

Expression of the B8.7 antigen on hairy cells and relation with the LMW-BCGF response.

Hairy cells are classified as B cell tumors at a preplasma cell stage of differentiation and are believed to represent cells undergoing a switch process. These cells are stimulated in vitro to DNA synthesis and multiplication in the presence of the lymphokine LMW-BCGF. We have tested the level of expression on these cells of the newly described B8.7 activation marker which has been reported to be associated with the capacity of various B cells to respond to LMW-BCGF. The presence of this marker has been readily detected on the hairy cells of 10 of the 12 patients tested in this study; interestingly, for one of the negative cases, the tumor cells were unable to proliferate in response to LMW-BCGF. As on normal B cells, a marked inhibition of the LMW-BCGF dependent response could be achieved in the presence of a monoclonal anti-B8.7 antibody, sustaining the proposal that the B8.7 molecule is involved in the signaling pathway of this growth factor. IFN-alpha is highly efficient in the therapy of hairy cell leukemia (HCL), and we confirm in the present study that IFN-alpha also inhibits the LMW-BCGF dependent proliferation of hairy cells in vitro. In addition, we show that this inhibition is independent of a significant modulation of the B8.7 antigen, a molecule putatively associated with the LMW-BCGF receptor.

HIV-associated endometritis.

Using immunohistochemical staining, in situ hybridization and a combination of both, we demonstrate here the replication of HIV in the endometrial stroma. Infected cells do not belong to the T-lymphocyte lineage but rather to a monocyte-macrophage cell type. This report suggests a possible relationship between HIV infection and endometritis. Moreover, HIV replication in endometrial tissues could play a role in heterosexual and materno-fetal transmission.

HIV-induced, HIV-specific in vitro antibody response by B-cells from HIV-seropositive individuals.

OBJECTIVE: Recent studies have shown that B-cells from HIV-infected patients can secrete anti-HIV antibodies in vitro and that they represent 20-40% of immunoglobulin (Ig)-secreting B-cells in vivo. This study was designed to investigate the precise role of HIV in this in vitro antibody production. DESIGN AND METHODS: B-cells from HIV-infected patients [asymptomatic, n = 28; symptomatic (AIDS), n = 14], from seronegative adult volunteers (n = 22) and subjects at high risk for HIV infection (n = 15) were cultured in vitro in the presence of pokeweed mitogen, Staphylococcus aureus cowan or HIV, and T-cells or interleukins (IL). Non-specific Ig production and specific anti-HIV antibody (Ab) production were measured by enzyme-linked immunosorbent and Western blot assays. RESULTS: We found that HIV induced a specific response in cultured B-cells from seropositive patients, in contrast with cultured B-cells from uninfected normal individuals. The characteristics of the HIV-induced response differed from those of a spontaneous or a mitogen-induced response. Anti-HIV Ab production was optimal on day 8-10, when B-cells were cultured with recombinant IL-2 and recombinant interferon-alpha in the presence of infectious virus or recombinant gp160 Env protein. The anti-HIV Ab were mainly directed against Env proteins. Interaction of HIV with B-cells involved surface IgG but not CD4 antigen. Autologous CD8+ T-cells had a non-specific inhibitory effect. Both CD5+ and CD5- B-cells produced anti-HIV Ab. No anti-HIV Ab production was observed in B-cells from high-risk HIV-seronegative individuals. CONCLUSION: HIV (infectious virus or gp160) can induce B-cells from infected patients to secrete specific anti-HIV Ab in vitro.

Activation of cytotoxic cells in hyperplastic lymph nodes from HIV-infected patients.

Serine esterase B (SE B) is a protein contained in cytoplasmic granules of cytotoxic T lymphocytes and natural killer cells; SE B gene is transcribed upon activation of these cytotoxic cells. In order to show the in vivo interactions between HIV-infected cells and anti-HIV cytotoxic cells we analysed, by in situ hybridization, the expression of the SE B gene in eight hyperplastic lymph nodes from HIV-1-infected patients presenting with persistent generalized lymphadenopathy. We detected numerous cells expressing the SE B gene. The mean number of positive cells was 3.2 times higher in HIV lymph nodes than in six non-HIV hyperplastic lymph nodes studied in parallel (P less than 0.05). In control lymph nodes, the SE B gene was expressed only in interfollicular areas; virtually no cells expressed the SE B gene within follicles. In contrast, in HIV lymph nodes cells expressing the SE B gene were distributed either in interfollicular areas or within follicles. Expression of the SE B gene inside follicles was thus a specific feature of HIV lymph nodes (P less than 0.001) and was associated with the presence of HIV antigens and RNA at the same site. These results suggest that cytotoxic cells are activated in follicles of HIV lymph nodes and may be involved in the lysis of HIV-infected cells. Such a phenomenon may explain the development of follicle lysis, a specific feature of HIV lymph nodes. It may also inhibit the spreading of HIV infection.

Syncytium induction by fresh HIV isolates: quantitative analysis using a transactivation beta-gal assay.

We used a quantitative bioassay (the beta-gal assay) to visualize and quantify syncytium induction by fresh HIV isolates. This bioassay is based on the transactivation by tat of a chimeric gene comprising an HIV-1 long terminal repeat (LTR) fused to a modified lacZ gene of Escherichia coli. The chimeric gene encodes a beta-galactosidase which is translocated to the nucleus. It allows the enzymatic staining of all nuclei from HIV-induced syncytia. Using this unequivocal assay (the beta-gal assay), we could assess the syncytium-inducing properties of fresh HIV isolates after only 4 days of coculture of patient lymphocytes with activated normal lymphocytes. Syncytium-inducing HIV isolates were detected in 11 out of 40 seropositive patients studied. They were isolated mainly from AIDS patients: eight out of 17 grade IV (according to Centers for Disease Control criteria) patients were infected with syncytium-inducing strains. However, of 23 grade II and III patients tested, syncytium-inducing HIV strains were isolated from three cases. These three patients displayed no detectable p24 antigenaemia and had a CD4+ cell count of greater than 300 cells/microliter. The in vitro replication rate of HIV grown from 36 patient blood samples was then examined by sequential p24 antigen measurements in coculture supernatants. The 10 samples leading to syncytium formation also exhibited the highest replication rate. The possibility of unequivocally detecting syncytium-inducing strains after only a few days of coculture will make this detection routine and rapid. In addition, the limited period of amplification required is a significant advantage as it minimizes the emergence of HIV variants selected during long-term in vitro cultures.

Acute upregulation of CCR-5 expression by CD4+ T lymphocytes in HIV-infected patients treated with interleukin-2. ANRS 048 IL-2 Study Group.

BACKGROUND: The treatment of HIV-infected patients with interleukin (IL)-2 causes a sustained increase in CD4+ T-lymphocyte counts, involving both naive and memory cells. However, the short-term immunological effects of IL-2, which may shed light on the mechanism of immune reconstitution by this cytokine, are unknown. OBJECTIVE: To evaluate the acute effect of IL-2 on circulating T-lymphocyte subpopulations and their expression of chemokine receptors. DESIGN AND METHODS: Flow cytometry, reverse transcriptase polymerase chain reaction and chemokine receptor function experiments were performed before and after 5 days of IL-2 administration in 30 HIV-infected patients. RESULTS: IL-2 induced an acute lymphopenia of both naive and memory T-helper (TH) lymphocytes. This was associated with a large increase in CC-chemokine receptor (CCR)-5 and CCR-2b expression by TH cells. Before IL-2 treatment, CCR-5 was mostly produced by CD62L- memory TH lymphocytes. After 5 days of IL-2 administration, the level of CCR-5 mRNA in circulating cells was 18.6 times higher than before treatment (P < 0.002). CCR-5 expression was upregulated in CD62L- memory TH lymphocytes, but also in CD62L+ memory and in naive (CD62L+ CD45RO-) TH lymphocytes. IL-2 treatment also increased the function of CCR-5 in TH cells. CONCLUSIONS: Chemokine receptors are involved in trafficking of lymphocytes. The IL-2-induced upregulation of chemokine receptors in TH cells may thus play a role in the acute effects of this cytokine in TH lymphocyte redistribution.

Early control of HIV replication in primary HIV-1 infection treated with antiretroviral drugs and pegylated IFN alpha: results from the Primoferon A (ANRS 086) Study.

IFN alpha has both antiviral and immunostimulating properties. The ANRS086 Primoferon A Study evaluated in 12 patients with primary HIV infection the tolerance and efficacy of an early and transient administration of pegylated IFN alpha, in addition to highly active antiretroviral therapy. Tolerance was good, and this regimen allowed the early control of HIV replication and rapid decay of the viral reservoir. These results support the initiation of comparative studies with pegylated INF alpha in primary HIV infection.

Paf-acether-induced superoxide anion generation in human B cell line.

Paf-acether (paf) and lyso phospholipids induced an oxydative burst on EBV-transformed B lymphocyte cell line. Superoxide anion formation measured by lucigenin-dependent chemiluminescence was dependent on both paf concentration and time-course of challenge. Paf C18:0 at 10 microM was more potent than its C16:0 analogue at the same concentration. Choline-containing phospholipids with 2-acyl (long chain) were inactive. The paf antagonists BN 52021 and WEB 2086 structurally unrelated to paf were inactive whereas paf structural analogue CV 3988 inhibited superoxide formation induced by paf and lysophospholipids. Such a phospholipid-induced oxydative burst in B cells might exert an effect in the numerous pathophysiological situations where large amounts of paf are produced by phagocytic cells.

IL4 and IL13 receptors share the gamma c chain and activate STAT6, STAT3 and STAT5 proteins in normal human B cells.

IL13 induces the same biological effects as IL4 in normal human B cells. We show that as in the IL4R complex, both IL4R alpha and IL2R gamma c are components of the IL13R and that both cytokines induced STAT6, STAT3 and STAT5 activation in B cells. In spite of this similar downstream signalling, IL4 and IL13 used a different set of Janus kinases: IL13 is unable to activate JAK1 and JAK3.

gp 140, the C3d/EBV receptor (CR2), is phosphorylated upon in vitro activation of human peripheral B lymphocytes.

gp 140, the C3d/EBV receptor (CR2), is a specific marker of human B lymphocytes. Very recent data suggest that CR2 is a membrane site involved in early B cell activation. These properties of CR2 led us to analyze the molecular events associated with gp 140. We analyzed whether in some conditions of B lymphocyte activation, CR2 could be phosphorylated. We have found that when highly enriched peripheral B cells were cultured for 48 h with anti-mu Ab and/or SAC, in order to provide an optimal activating signal, phosphorylation of the CR2 was induced.

Mechanisms of opsonized HIV entry in normal B lymphocytes.

Using our in vitro model of normal B cell infection that functions with low doses of HIV but requires virus opsonization by seropositive patient serum, and complement, we analyzed what receptors allowed virus entry. Here, we show that HIV infection of B cells occurs through 2 major receptors: the CD4 antigen and the CR1/CR2 complex. These 2 pathways work independently since a complete inhibition of virus entry requires both CD4 and CD21/CD35 blockade on CD4dim tonsillar B cells whereas only the latter is critical on CD4-negative B cells.

Admission criteria for immunogerontological studies in man: the SENIEUR protocol.

Immunogerontological studies in man have often led to conflicting results. One of the main reasons is the selection of the subjects to be studied. Admission criteria such as "apparently healthy" or "without overt disease" seem insufficient to exclude underlying disease which might influence the immune system and thereby the results. In an attempt to solve this problem, the SENIEUR protocol described in this paper was developed by a working party in the framework of the EURAGE Concerted Action Programme on Ageing of the European Community. This protocol establishes strict admission criteria for immunogerontological studies in man based on clinical information and laboratory data, and it sets limits to pharmacological interference. The use of this protocol will lead to standardization between centers and also to a closer study of the influence of age as such on the immune system. These findings in the immunologically "optimally aged" can also serve as reference values for immunogerontological studies in subjects who do not meet the SENIEUR criteria. In this way the use of this protocol can contribute to the dissection of the influence of disease versus ageing on the immune system.