Search

Wegener's granulomatosis (WG) is an inflammatory, destructive, angiotropic lesion. The inflammatory process involves accumulation of macrophages, lymphocytes, and polymorphonuclear neutrophils. We studied 6 lung biopsy specimens from patients with WG to characterize the cellular infiltrate and to analyze the mechanism of immune cell recruitment. We show that lymphocytes accumulating in WG lesions are mostly memory CD4(+)CD45RO(+) T lymphocytes and, although less numerous, CD8(+)CD45RO(+) T lymphocytes. Few if any B lymphocytes or natural killer cells are present within lesions. The chemokine RANTES (regulated upon activation in normal T cells, expressed and secreted) has been reported to recruit memory T lymphocytes and macrophages selectively. We used reverse-transcription polymerase chain reaction, in situ hybridization, and immunohistochemistry to study its production in WG. RANTES was expressed at a higher level in WG lungs than in normal controls, especially around microabscesses. As visualized immunohistochemically in serial sections with anti-RANTES monoclonal antibody, RANTES production was produced mainly by macrophages. Expression of the gene coding for interferon-gamma (IFN-gamma), a potent RANTES inducer, was also studied. Its expression was also much stronger in WG than in controls. Our observations are consistent with a cascade of events leading to the recruitment of immune cells in WG, sequentially involving production of IFN-gamma by T lymphocytes and RANTES production by macrophages, leading to the homing of memory T-helper lymphocytes and macrophages. HUM PATHOL 32:320-326., (Copyright 2001 by W.B. Saunders Company)

Increase Th2 cytokine production may contribute to some clinical manifestations of HIV infection, and studies have suggested that IL-13 rather than IL-4 is involved in these conditions. We directly tested this hypothesis by administrating IL-13 to SIV-infected macaques. SIV-infected rhesus macaques received a daily subcutaneous injection for 21 days of either IL-13 (10 microg/kg/day) or a placebo. The four macaques treated with IL-13 experienced body weight loss (9.95 +/- 0.71%) related to intestinal tract damage: they all suffered from a complete atrophy of duodenal villi. This was presumably due to premature epithelial cell death: proliferating Ki67+ cells in glandular crypts were as numerous as in control animals, but many epithelial cells developed apoptosis. The duodenal mucosa was infiltrated with cells expressing CD56 and PEN5, two markers of NK cells, and there was a deregulation of local cytokine and chemokine production characterized by a decrease in IL-10 gene expression (25% of controls) and an increase in gene expression for IFN-gamma (4-fold control), MIP-1alpha (8-fold control), and MIP-1beta (13-fold control). Thus, IL-13 can induce digestive epithelial cell injury in vivo in primates infected with a retrovirus. Therefore, its role should be considered in digestive manifestations of HIV infection as well as in other disorders associated with intestinal epithelial atrophy.

We analyzed the production and the roles of IL-6, IL-10, and IL-13 in B-lymphoid malignancies and in specific diseases with B-lymphocyte hyperactivity. Both IL-13 and IL-10 genes are expressed in B-cell lymphomas. However, their contribution to tumor progression is unclear. In certain lymphoproliferative disorders that develop in transplanted patients, IL-6 is produced by malignant cells and is a major factor of their proliferation. In other lymphomas, the IL-6 gene is expressed only in malignancies where differentiated malignant cells are present. In these lymphomas, IL-6 is produced by stromal cells, and the malignant cells express the IL-6 receptor. In patients with HIV infection, the level of production of IL-6, IL-10, and IL-13 is not higher than those of other conditions with immune activation. However, IL-6 contributes to increased production of IgG and IgA in vivo. In Castleman's disease, IL-6 is produced in the lymph node germinal centers, partly originating from follicular dendritic cells, which may explain some of the pathogenesis of this disease. In systemic lupus erythematosus, the critical cytokine is IL-10, which is produced in large amounts by B lymphocytes and monocytes and is responsible for autoantibody production. Taken together, these data emphasize the roles of IL-6 and IL-10, usually produced by nonlymphoid cells, on B lymphocytes, either malignant or hyperactivated.

Objective: Polymyositis (PM) and cermatomyositis (DM) are inflammatory muscle diseases of autoimmune origin. A chronic mononuclear cell infiltrate is always present around PM and DM muscle damage. In PM, the predominant cells are activated cytotoxic cells, natural killer, and CD8+ T lymphocytes. Cytotoxic cells can kill the target cells via 2 mechanisms. Both pathways induce target cell death by releasing the molecules (granule exocytosis) perforin (PF), which attacks the target cell membrane and causes cell death by necrosis, and TIA-1 protein and granzyme-B (GZB), possibly responsible for apoptosis. We studied the mechanisms of lysis in muscle biopsies of myositis., Methods: We used a panel of monoclonal antibodies to determine the phenotypes of reactive lymphocyte subsets, in situ hybridization to study the expression of PF and GZB genes, and immunohistochemistry to evaluate TIA-1 protein production in muscle biopsies from 14 patients with myositis (11 PM/3 DM). Results were compared to those obtained from muscle biopsies of 12 control patients with muscle weakness, including patients with muscle dystrophy and vasculitis, but without myositis., Results: Abundant CD8+ cells, especially in endomysial sites in PM, formed the predominant phenotype. The predominant mononuclear cells observed in DM were CD4+ T cells and CD22+ B lymphocytes, in perivascular sites. The GZB and PF genes and the TIA-1 protein were expressed simultaneously in muscle samples from patients with myositis. GZB, PF, and TIA-1 positive cells were predominantly located in endomysial sites of PM, in the nonnecrotic muscle fibers. In DM, these positive cells were rare., Conclusion: In myositis, especially PM, cytotoxic cells may cause muscle damage and muscle cell necrosis and/or apoptosis by releasing several proteins (PF, GZB, and TIA-1 proteins) responsible for the lysis of these stimulating target cells. Some drugs (prednisone and cyclosporine) inhibit the release of GZB and PF. Their efficacy may be due in part to this inhibitory effect.

The incidence of lymphomas is unusually high in human immunodeficiency virus (HIV)-infected patients. Because cytotoxic T lymphocytes (CTL) represent a major mechanism of the antitumoral immune response in immunocompetent individuals, we asked whether intratumoral activation of CTL was impaired in acquired immune deficiency syndrome (AIDS) lymphomas. Immunohistochemical experiments showed that in AIDS lymphomas intratumoral CD8-positive T lymphocytes accumulated and expressed the TIA-1 antigen, a marker of cytotoxic cells. Flow cytometry studies and in situ hybridization of lymphomatous tissue confirmed the differentiation of CD8-positive cells in cytotoxic cells and their activation, as assessed by their expression of CD38 and human leukocyte antigen (HLA) DR markers as well as the perforin and granzyme B genes, which code for two molecules involved in target cell killing. On average, perforin-producing cells were as numerous in AIDS lymphomas (5,647 +/- 2,655 cells/cm2) as in lymphomas from immunocompetent individuals (3,294 +/- 1,544 cells/cm2). The density of activated CD8-positive cells in the 22 AIDS lymphomas tested was not correlated with peripheral CD4-positive cell counts. These results suggest that in AIDS lymphomas the steps of differentiation and activation of cytotoxic CD8-positive cells are not altered by immune deficiency and that they can take place through pathways relatively independent of CD4-positive T lymphocytes. Thus, other mechanisms of immune deficiency should account for the increased frequency of lymphomas in patients with AIDS.

Expression of the IL-13 gene in malignant tissues from 26 human B-cell lymphoid malignancies was analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR). A positive signal was detected in 16 cases, which included high grade B lymphomas, follicular lymphomas and B-cell chronic lymphocytic leukemias. IL-13 mRNA was also detected in the 9 malignant B cell lines and in the 6 lymphoblastoid cell lines tested, as well as in freshly isolated malignant B cells from 2 patients with a Burkitt's lymphoma. Two of 8 T-cell lymphomas and 2 of 4 T-cell lines expressed the IL-13 gene. In contrast, IL-13 gene expression was not detected in any of the 5 non-lymphoid cell lines tested. No specific binding of radiolabeled IL-13 was detected on B cell lines, suggesting an absence of IL-13 receptors on such cells. This conclusion was also supported by the inability of IL-13 or anti-IL-13 antibodies to affect the growth of malignant B cells. Taken together, these results show that both malignant and EBV-transformed B lymphocytes, either freshly isolated or maintained as cell lines, express the IL-13 gene. This raises the question of the role of B lymphocyte-derived IL-13, a B lymphocyte stimulating cytokine, on the in vivo function of normal B lymphocytes as well as on the in vivo behaviour of B lymphoid malignancies.

To understand the selective accumulation of memory T helper lymphocytes and of macrophages in delayed-type hypersensitivity (DTH) granulomas, we studied the in situ production of RANTES, a chemokine initially characterized on the basis of its in vitro chemotactic properties for each of these cell populations. RANTES gene expression was studied by in situ hybridization in 15 human lymph nodes presenting typical DTH lesions related to either sarcoidosis or tuberculosis. A positive signal was detected in all cases. Labeling was specific for the DTH lesions, as very few if any positive cells were detected in the normal residual lymphoid tissue surrounding them or in reactive lymph nodes involved in a B lymphocyte response. RANTES gene expression was associated with the production of the protein, which was detected by immunochemistry in DTH lymph nodes. The morphological characteristics and distribution of positive cells in in situ hybridization and immunochemical experiments indicated that macrophages and endothelial cells, two cell populations not previously reported to produce RANTES, contributed to its production in DTH reactions. The ability of macrophages and endothelial cells to produce RANTES was confirmed by in vitro studies with alveolar macrophages and umbilical vein endothelial cells. In view of the chemotactic properties of RANTES for a limited range of cell populations, these results suggest that RANTES production in DTH granulomas may play a role in the selective accumulation of macrophages and memory T helper lymphocytes characterizing this type of cell-mediated immune reaction, and that macrophages and endothelial cells are involved in this production.

Human immunodeficiency virus infection leads to a deregulated production of a number of cytokines. Some of them (IL-1, IL-6, TNF-alpha, interferon-gamma) are produced in increased amounts in vivo, whereas the production of IL-2 is decreased. This latter abnormality plays a pivotal role in the establishment of the immunodeficiency. Some cytokines (IL-1, IL-6, TNF-alpha) stimulate the in vitro replication of HIV, whereas others (mainly the interferons) inhibit it. The effect of cytokines in vivo in the spreading of HIV remains, however, largely unknown. Cytokines may also be involved in the development of many clinical manifestations associated with HIV infection. IL-1, IL-6 and TNF-alpha may play a role in tissue damages associated with opportunistic infections, in HIV-related encephalopathy and in cachexia. Cytokines, mainly IL-6, IL-10 and IL-13, may stimulate the growth of malignant cells during Kaposi sarcoma or lymphomas. Better knowledge of the role of cytokines during HIV infection should allow new therapeutic approaches based on the use of either recombinant cytokines or specific antagonists, with the aim of limiting both HIV spreading and the clinical manifestations of this infection.

IL-6 serum levels are increased in patients suffering from GCA, and this cytokine may play a role in the systemic symptoms associated with this disease. We analyzed by in situ hybridization and immunohistochemistry whether IL-6 production originates from arterial granulomas in GCA. Seven arterial biopsy specimens taken from patients with histologically proved GCA were studied. IL-6 production was detected in all cases at both the mRNA and the protein levels. IL-6-producing cells were distributed in the intima, in the media, and in the adventitia. Positive cells were clearly enriched in the media, however, and particularly in contact with the internal elastic lamina. The morphology of the IL-6-producing cells showed that they belonged to several cell populations. In the media, most IL-6-producing cells were macrophages, whereas fibroblasts also produced IL-6 in the intima. Neither endothelial cells nor giant cells were found to express the IL-6 gene. Increased IL-6 serum concentrations returned to normal levels in most patients after administration of corticosteroids, indicating that inhibition of IL-6 production by GCA granuloma cells may be one of the mechanisms of action of corticosteroids in this condition. Production of IL-6 in abnormal arteries may thus participate to the systemic manifestations of GCA.

Interleukin-6 is a major B lymphocyte growth factor, and may play a role in the proliferation of malignant B lymphocytes. In order to provide arguments supporting such a role, the intratumoral production of IL-6 was studied by in situ hybridization and immunohistochemistry in 53 neoplastic tissues from B cell chronic lymphocytic leukemia or B lymphomas. IL-6-producing cells were detected in all samples but 5. However, the number of IL-6 producing cells was variable amongst the different cases. Increased density of IL-6-producing cells was highly dependent on the presence of malignant immunoblasts within the neoplastic clone. IL-6 was produced in a paracrine way, macrophages and endothelial cells being the main producers of the cytokine while malignant immunoblasts expressed the IL-6 receptor. Taken together, these results suggest that IL-6 may indeed act as a growth factor for malignant cells in some B lymphoproliferations and that this paracrine loop could be the target of new therapeutic approaches.

Fourteen monoclonal antibodies were used to immunohistochemically label 22 temporal artery biopsy specimens taken from patients with temporal arteritis before treatment (n = 10), after 1.3 days of corticotherapy (n = 6) and after 12-30 days of steroids (n = 6). Histological sections from untreated patients revealed an inflammatory infiltrate comprised of approximately equal proportions of macrophages and T lymphocytes; the majority of the latter belonged to the CD4+ subset (the CD4+/CD8+ ratio varied from 2/1 to 4/1, depending upon the biopsy). These cells expressed high levels of HLA DR and low levels of interleukin-2 (IL2) receptors. A few B lymphocytes and plasmocytes were seen, mainly in the adventitia. Antigen-presenting cells (APC) were always found in the damaged media and natural killer cells (few in number) were sometimes present. Some macrophages were positively immunolabeled for IL6. A short, 1-to-3-day course of corticosteroids did not appreciably modify the lesions: cells remained highly activated, APC were seen in half the biopsies and IL6 immunolabeling persisted. The findings were essentially the same in treated but poorly controlled patients. Biopsies from 2 patients in clinical and biological remission revealed the persistence of an active immunological process. These observations indicate that the immunological process is poorly controlled by corticosteroid therapy.

Expression of the interleukin (IL)-10/BCRF1 gene was studied by in situ hybridization in tissue samples from acquired immunodeficiency syndrome (AIDS) lymphomas using a BCRF1 probe which also recognizes the human IL-10 sequence. Hybridization was detected in 8 out of 15 lymphomas. In contrast, the IL-10/BCRF1 gene expression was detected in only 1 out of 11 lymphomas from human immunodeficiency virus (HIV)-seronegative patients (p = 0.05). In AIDS lymphomas, the number of cells labeled with a BCRF1-specific probe was dramatically lower than that of cells labeled with the IL-10/BCRF1 probe. Thus, the IL-10 rather than the BCRF1 gene was expressed. Production of IL-10 was associated with that of IL-10 mRNA, as shown by immunodetection of the protein in numerous cells. In contrast, BCRF1-producing cells were rarely detected. Both in situ hybridization and immunochemical experiments indicated that malignant cells were involved in this IL-10 synthesis. IL-10 production in AIDS lymphomas was associated with the presence of Epstein-Barr virus (EBV) in lymphomatous cells (p = 0.02). As IL-10 is a potent growth factor for human B lymphocytes, these results suggest that IL-10 may stimulate the proliferation of malignant cells in an autocrine pathway in a number of AIDS lymphomas, and that EBV and HIV may synergistically trigger its production.

We recently demonstrated that IL-2 is produced by reactive T cells in CD25-positive malignant lymphomas (ML). Using in situ hybridization, we investigated IL-6 mRNA expression in these CD25-positive ML. The ML tested included 9 anaplastic large cell lymphomas and 3 B-diffuse large cell lymphomas. Five CD25-negative ML were studied as controls. We show that IL-6 producing cells are present in all these ML. The density of positive cells was heterogeneous from case to case. However 3 cases of CD25-positive ML showed a dramatically higher density of IL-6 producing cells (70, 50, 43 producing cells per 10,000 cells, respectively) as compared to the other 9 cases of CD25-positive ML (mean 6.03 +/- 2.1 per 10,000). Morphological and topographical data suggested that several types of cells including fibroblasts, lymphocytes, macrophages and endothelial cells may synthesize IL-6. A combination of immunohistochemistry and in situ hybridization showed that reactive T cells and endothelial cells express the IL-6 gene whereas CD30-positive ML cells do not express this gene. Previous studies showed that IL-6 was capable to induce IL-2 receptor expression as well as production of IL-2 and stimulation of lymphomatous cells growth. Our present results indicate that the paracrine production of this cytokine may play a role in the proliferation of malignant lymphomas.

Sarcoidosis is a chronic granulomatous disease that may be considered to be a human model for the delayed-type hypersensitivity reaction. The expression of cytokine genes in organs displaying sarcoid granulomas was analyzed by in situ hybridization with several cytokine probes using biopsies from 11 sarcoid lymph nodes. We detected cells expressing interleukin-1 beta (IL-1 beta), tumor necrosis factor-alpha (TNF-alpha), IL-6, IL-2, and interferon-gamma (IFN-gamma) genes in all lymph nodes. The major finding of this study was that cytokine genes are independently expressed. Of the monokine genes, the IL-1 beta gene was preferentially expressed. The distribution of cells containing IL-1 beta mRNA was characterized by their amalgamation in clusters inside sarcoid granulomas. Cells expressing the TNF-alpha gene were located exclusively inside granulomas and were always scattered. Cells expressing the IL-6 gene or the IL-1 alpha gene were found scattered inside sarcoid granulomas and in the residual lymphoid tissue. The number of cells expressing the IL-1 beta gene was significantly higher than that of cells expressing TNF-alpha gene (P = .001), IL-6 gene (P = .007), or IL-1 alpha gene (P less than .001). Of the cells expressing lymphokine genes, those expressing the IFN-gamma gene were 31.9 (+/- 7.6) times more frequent than those expressing the IL-2 gene (P less than .001). Cells containing IFN-gamma mRNA were detected mainly inside sarcoid granulomas, whereas cells containing IL-2 mRNA were randomly distributed. These results show that each monokine gene or lymphokine gene can be independently expressed in vivo. The high expression level of the IL-1 beta gene and the IFN-gamma gene inside granulomas may be specific to delayed-type hypersensitivity immune reactions.

Monokines play a major role in the regulation of hepatocyte functions. To document a possible in situ production of these mediators under physiological conditions, expression of IL-1 beta and of IL-6 genes was analyzed by in situ hybridization in four histologically normal human livers. We detected cells containing IL-1 beta mRNA or IL-6 mRNA in all cases. Cells expressing either the IL-1 beta gene or the IL-6 gene were found with equal frequency and were similarly distributed. Although present in all liver compartments, they were selectively enriched in portal areas, in which they were detected both in endothelial positions and in perivascular connective tissues. Few positive cells were observed in hepatic lobules, most of them being located in the walls of centrolobular veins, in an endothelial position. Subcapsular cells were also shown to express monokine genes. The location of positive cells and their pattern of labelling suggested that macrophages, fibroblasts and endothelial cells were the main cell populations expressing monokine genes. In contrast, Kupffer cells, biliary epithelial cells and hepatocytes did not express monokine genes. No marker of immune activation other than monokine gene expression was detected in these histologically normal livers. The expression of the IL-1 beta gene and of the IL-6 gene may be induced by gut-derived LPS, and could play a role in the modulation of hepatocyte function in normal liver.

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.

We analyzed by in situ hybridization the expression of four interleukin genes (interleukin-beta [IL-1 beta], IL-6, IL-2, and interferon-gamma) in seven thymuses displaying a follicular hyperplasia. The seven thymuses were obtained from patients with myasthenia gravis. Interleukin-1 beta- and IL-6-producing cells were detected in similar amounts and with similar distributions: mainly in perifollicular areas and in the connective structures emerging from the septae at the site of cortex disruption. The comparison of in situ hybridization and immunohistochemical results suggested that thymic epithelial cells and/or perifollicular macrophages were responsible for this production. Interleukin-2-producing cells were detected in perifollicular areas and, to a lesser extent, inside follicles. They were clearly outnumbered by CD25-positive cells which were similarly distributed. Despite the expression of these molecular and immunohistochemical markers of T-cell activation, interferon-gamma-producing cells were extremely rare in myasthenic thymuses. The pattern of interleukin production (which was virtually absent in normal control thymuses) in myasthenic thymuses was different from that in benign hyperplastic lymph nodes. This interleukin production may play a role in the development of follicular hyperplasia in myasthenic thymuses, a phenomenon which is associated with the in situ production of autoantibodies.

Macrophage infiltration is a constant feature of human virus-infected tissues. However, the in situ functional status of these cells remains undetermined. In order to document an activation of macrophages in virus-infected tissues, the expression of IL-1 beta and IL-6 genes was analyzed using in situ hybridization. Several tissues were studied, as well as infections induced by different viruses: lymph nodes infected by HIV-1 (9 cases) or EBV (one case), lungs infected by CMV (5 cases) or adenovirus (1 case), livers infected by HBV, either chronically (2 cases) or acutely (7 cases presenting a fulminant hepatitis). With the exception of fulminant HBV hepatitis, IL-1 beta and IL-6 genes were expressed in all cases. IL-1 beta and IL-6 genes were usually coordinately regulated, as cells containing IL-1 beta or IL-6 mRNA were present in identical amounts and displayed a similar distribution. Analysis of the location and the morphology of monokine gene-expressing cells indicated that both small macrophages and endothelial cells expressed IL-1 beta and IL-6 genes. However, neither tingible body macrophages present in lymph node follicles nor Kupffer cells expressed these genes at a detectable level. Infected cells themselves were also negative for monokine gene expression. These findings indicate that expression of IL-1 beta and IL-6 genes by reactive cells may play a role in viral spreading limitation as well as virus-induced tissue damage.

We used in situ hybridization to study the expression of interleukin genes in sarcoidosis and in persistent generalized lymphadenopathy of HIV disease. In both cases, we found a dramatic over-expression of the interferon-gamma (IFN gamma) gene as compared to that of the interleukin-2 (IL-2) gene. In sarcoidosis, IFN gamma producing cells are CD4 T cells and are associated with IL-1 beta gene expressing monocytic cells. In HIV lymphadenopathy IFN gamma producing cells are C8 T cells engaged in cytotoxic function, as evidenced by the concomitant expression of serine esterase B gene. Thus distinct patterns of interleukin production can be defined in vivo in selected immunopathological situations.

In situ hybridization with specific RNA radiolabeled probes was used to analyze the production of interleukin-2 (IL-2) and interferon-gamma (IFN-gamma) by 21 low-grade follicular lymphomas (FLs). All of the 21 FLs tested contained lymphokine-synthesizing cells in the interfollicular and follicular areas. Enumeration of lymphokine synthesizing cells indicated heterogeneous IL-2 production among lymph nodes tested; 2 of the 21 had much higher densities of IL-2-producing cells (855 and 570/cm2) than did the remaining 19 (mean, 92 +/- 15/cm2). IFN-gamma-producing cells displayed no such variation (mean, 77 +/- 8 IFN-gamma-producing cells/cm2). The mean IL-2/IFN-gamma-producing cell ratio was 2.69 +/- 0.84, indicating preferential induction of IL-2. The detailed distribution of lymphokine-producing cells showed that IL-2 and IFN-gamma-producing cells were located mainly in the follicular areas. The mean follicular/interfollicular ratio was 1.82 +/- 0.16 for IL-2 and 1.92 +/- 0.19 for IFN-gamma-producing cells. The results show that T-cell activation, defined by lymphokine production, occurs in FL lymph nodes in direct contact with malignant B cells. Thus, lymphokine production may play an important role in the control of tumor growth, which is the result of interaction between tumor cells and host-derived immune reaction.

Cystic fibrosis is associated with an cAMP-regulated channel defect, which has been evidenced in many cell types including B lymphocytes. To document a B-cell dysfunction potentially related to this defect, we studied the in vitro IgG production by lymphocytes from 11 cystic fibrosis patients. B lymphocytes were co-cultured with autologous monocytes and stimulated with Staphylococcus aureus Cowan or with Nocardia-delipidated cell mitogen in the presence of low concentrations of IL2. Cystic fibrosis patients' cells produced amounts of IgG comparable with that of normal and control patients' cells. However, dexamethasone (10(-7) mol l-1) had no effect on the response of cystic fibrosis patients' cells, whereas it enhanced that of the latter two groups. This resistance of cystic fibrosis cells was true with concentrations of dexamethasone up to 10(-6) mol l-1, whereas this agent induced a dose-related enhancement from 10(-8) to 10(-6) mol l-1 in cultures of normal cells. Co-culture experiments showed that cystic fibrosis B lymphocytes themselves are resistant to the effect of dexamethasone. In contrast dexamethasone normally suppressed the anti-CD3 antibody-induced response of cystic fibrosis T cells in the presence of IL2 and the IL1 alpha- or beta-induced collagenase production of cystic fibrosis fibroblast cell lines. Thus cystic fibrosis B lymphocytes exhibit a selective defect which may interfere with the normal interactions between the hormonal and immune systems and may participate in the sensitivity of cystic fibrosis patients to bacterial bronchopulmonary infections.

We analyzed the effects of interleukin 2 (IL 2) and IL 4 isolated and in association on the specific response of human B cells triggered by trinitrophenylated polyacrylamide beads (TNP-PAA). IL 2 induced an increase (more than 10 times) in the number of hapten-binding cells [detected by a rosette-forming cell (RFC) assay] as well as the generation of antibody-producing cells [detected by a plaque-forming cell (PFC) assay]. IL 4 induced an isolated RFC response without PFC response. We verified that the IL 4 (as well as 12)-induced RFC were hapten specific and mediated through membrane IgM. Density fractionation experiments showed that IL 4-induced RFC were equally distributed between high-density and intermediate-density B cells. IL 2 appeared to drive more B cells into the intermediate density fraction. IL 2-induced PFC belonged to the RFC population and were intermediate-size B cells. IL 2 drove more RFC into an activated stage and it induced the differentiation of a number of them into antibody-producing cells. The evaluation of the proportion of RFC able to incorporate thymidine showed that both IL induced a substantial proliferation of antigen-activated B cells. However, IL 4 inhibited the IL 2-dependent PFC without affecting the number of RFC nor the proportion of proliferating RFC induced by this IL. These results directly demonstrate that human IL 4 triggers the expansion of antigen-activated B cells and selectively inhibits the IL 2-induced differentiation.

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.

Expression of the IL-2 receptor (Tac antigen/CD25) is documented in malignant lymphomas. Because IL-2 is a major lymphocyte growth factor, an IL-2-dependent growth could be involved in the proliferation of Tac-positive lymphomas. Indeed such a mechanism has been demonstrated experimentally for the growth of T-cell lines. To investigate this point in human lymphomas, we used in situ hybridization to analyze the expression of the IL-2 gene in 20 non-Hodgkin's lymphomas, among which 12 expressed the IL-2 receptor. Nine of these were anaplastic large cell lymphomas expressing the Ki-1-related antigen. We here show that IL-2-producing cells are present in all the lymphomas we analyzed. As a mean, there is no significant difference in the percentage of IL-2-producing cells between Tac-positive and -negative lymphomas. However, the level of IL-2 production is highly heterogeneous in both groups, and the highest density of IL-2-producing cells was observed in 2 Tac-positive lymphomas. Simultaneous detection of cellular antigens and of IL-2 mRNA demonstrates that IL-2 is produced by reactive T cells rather than by tumor cells. These results suggest that if IL-2 is involved in the growth of Tac-positive lymphomas, it acts as a paracrine, rather than an autocrine, factor.

The in vitro antibody response to most T-independent antigens is inhibited by Azathioprine (Az) only at concentrations of 1-10 mug/ml. In contrast, B cell response to T-dependent antigens and to the T-independent antigen TNP-Polyacrylamide is sensitive to low Az concentrations (10(-2) mug/ml). These data suggest the existence of two different B cell activation processes : a. The activation by B cell mitogens or T-independent antigens with a mitogenic moiety which are Az-resistant; b. The activation by T-dependent antigens or T-independent antigens without a mitogenic moiety which are Az-sensitive.

The effect of exogeneous prostaglandin E2 (PGE2) on the specific plaque-forming cell (PFC) response in cultures of non-adherent human peripheral blood mononuclear cells (PBM) was tested. When added on Day 2 of the cultures PGE2 inhibits the induction of the PFC response, and the maximum inhibition (50%) is obtained with 300 nM PGE2. When PBM are cultured during the first 24 hr with the same concentration of PGE2 their PFC response is enhanced and the target of this enhancement is a T cell. When PGE2 is added on Day 0 it does not affect the response, probably because of a balance between these two opposing effects. However, in the latter conditions a prostaglandin-free monocyte supernatant can render PGE2 suppressive. The monocyte supernatant acts by inhibiting the stimulatory effect after the interaction between PGE2 and T cells. Thus the effect of PGE2 depends on its time of action and on the concomitant production of a nondialyzable factor by monocytes.

This work was designed to delineate the anti-hapten antibody (Ab) response induced by trinitrophenol-polyacrylamide (TNP-PAA) beads from the nonspecific B-cell response which concomitantly occurs in human peripheral blood mononuclear cell (PBMC) cultures. Indeed human PBMC produce consistent amounts of immunoglobulins when cultured at high cell density in the presence of fetal bovine serum, regardless of the presence of antigen. In contrast, the stimulation of such cultures by TNP-PAA leads to an Ab response characterized by the following: cells secreting anti-hapten Ab at a high rate (detected by a plaque-forming cel (PFC) assay); a 10-30 times enhancement in the number of hapten-specific binding cells (detected by a rosette-forming cell (RFC) assay); the production of anti-TNP IgM Ab (detected by an ELISA assay). The anti-TNP response is specifically triggered by the particulate antigen, as shown by the following: The TNP-PAA antigen induces a clear-cut increase in the amount of anti-TNP Ab whereas it only marginally increases that of total IgM. The anti-TNP Ab response is specifically abolished when anti-TNP RFC are depleted from the PBMC preparation before the initiation of the cultures. The anti-TNP Ab response is specifically abolished when PBMC are triggered by TNP-PAA in the concomitant presence of a soluble TNP-protein conjugate. These results demonstrate the ability of polymeric antigens to specifically activate human peripheral blood B cells.

The specific response of human peripheral blood lymphocyte cultures to TNP-polyacrylamide was suppressed by the addition of concanavalin A (Con A). A dose of Con A (0.5 microgram/ml) could be selected, which induced a reproducible but incomplete suppression independent of the magnitude of the anti-TNP response. Con A-stimulated cells could transfer the suppression to autologous or allogeneic responding cells. The suppressor activity was present in the E-rosette forming cell fraction and was abolished by mitomycin C treatment prior to incubation. With a particular batch of foetal bovine serum, spontaneous suppressor cells were produced which suppressed the response of autologous and allogeneic lymphocytes. In allogenic mixtures, the otherwise enhancing allogeneic effect was replaced by a marked suppression from spontaneous suppressor cells. This suppression was higher than that exerted on autologous lymphocytes, suggesting that an unexpected negative allogenic effect had taken place. Spontaneous suppressors were ineffective when added on day 2 of a culture responding to TNP-polyacrylamide, whereas Con A induced suppressors were fully effective.

The C3d receptor is a specific marker of B lymphocytes. Recently we have shown that C3d receptor activity is carried by a gp 140 membrane antigen. A polyclonal antibody has been prepared by immunizing a rabbit with highly purified gp 140 molecule isolated from membranes of the human B lymphoblastoid cell line Raji and its high specificity was previously demonstrated. We tested the effect of this antibody to the C3d receptor on the B cell proliferative response. Purified B cells from human blood were induced to proliferate by a B cell growth factor (BCGF)-containing partially purified supernatant from activated T cells. The anti-C3d receptor F(ab')2 enhanced the BCGF-dependent B cell proliferation. This effect was dose dependent, was observed in the presence of different concentrations of BCGF and did not correspond to a change in the time course of the response. The anti-C3d receptor F(ab')2 had no mitogenic effect in the absence of T cell supernatant. In contrast the undigested anti-C3d receptor IgG suppressed the BCGF-dependent B cell proliferation. These results emphasize the potentialities of anti-gp 140 F(ab')2 to explore the involvement of the C3d receptor in the regulation of B cell response to T cell products.

Nonspecific T cell factors produced by lectin-activated human peripheral blood lymphocytes (PBL) were used to restore the T-dependent B cell response to trinitrophenyl-polyacrylamide (TNP-PAA). Preincubation experiments with the particulate antigen TNP-PAA and/or a soluble TNP-protein conjugate show that a first specific signal provided by the antigen and nonspecific lymphokines sequentially acts on B cells. By gel filtration the T cell-replacing factor (TRF) activity is present in the 30-15-kDa fraction of T cell supernatants and is associated to interleukin 2 (IL2). However, absorption of IL2 does not abolish the TRF activity. Moreover, chromatofocusing of this 30-15-kDa material allows the obtaining of an IL2-free fraction containing a differentiation factor (with an isoelectric point of 5.7 +/- 0.2). The ability of this fraction to restore the anti-TNP response is manifest in the presence of a 50-kDa B cell growth factor. This latter, prepared by a combination of absorption on concanavalin A-Sepharose and gel filtration, was IL2 free and unable to support the anti-TNP response. We thus directly demonstrate that in the absence of IL2 three separate signals (the antigen, T cell-derived growth and differentiation factors) are involved in human-specific B cell response.

The specific plaque-forming cell response induced by trinitrophenyl polyacrylamide beads in cultures of nonadherent human peripheral blood mononuclear cells (PBM) is resistant to the inhibitory effect of hydrocortisone (HC). Previous results showed that low concentrations (up to 10(-7 M) of HC inhibit the same response in cultures of unfractionated human PBM. It is shown that the addition of 5-10% monocytes to nonadherent PBM renders their response HC-sensitive. One mechanism of the interaction between HC and monocytes is the potentiation of prostaglandin E2-mediated suppression by HC, which can be demonstrated in vitro and after in vivo administration of HC. However, interaction with a non-prostaglandin-mediated suppression by monocytes should be involved, particularly with high (10(-6) M) concentrations of HC.

Upon in vitro activation by Staphylococcus aureus Cowan I strain (SAC) human peripheral blood B cells produce only marginal amounts of Ig when cultured in the presence of interleukin 2 (IL2; 10 U/ml). This response is only moderately increased by the addition of monocytes or of IL1. In the presence of dexamethasone (DM; 10(-7)-10(-8) M) microgram amounts of both IgM and IgG are produced in co-cultures of B cells and monocytes. This response is not modified by inhibitors of cyclooxygenase and is specifically inhibited by a monoclonal antibody interfering with the binding of IL2 to its receptor. This enhancing effect of DM is not observed in the absence of monocytes even if IL1 is added to the cultures. Moreover, monocytes pretreated with DM stimulate the response of B cells cultures in the absence of DM. Enhancement of Ig production by DM and monocytes could be demonstrated with B cells obtained from a patient suffering from a hyperlymphocytic form of B cell type chronic lymphocytic leukemia, and in this case only IgM was produced. Importantly, DM fully inhibited the IL2-dependent proliferation of these monoclonal B cells. Thus, physiological concentrations of DM can modulate monocytic function to enhance the differentiative effect of IL2.

The effect of azathioprine (Az) on the primary in vitro antibody response of mouse spleen cell cultures has been studied. The response towards T cell-dependent antigens is suppressed by low Az concentrations (50% inhibition by 10(-2) mug/ml and 100% suppression by 10(-1) mug/ml). The same pattern is observed when Az addition is delayed until day 2, but the suppression is absent or partial when Az is added on day 3. In the early period (day 0 to day 1) the effect of Az is reversible upon addition of an excess of purine nucleosides. In contrast, the response to a T cell-independent antigen (TNP-T4) is relatively insensitive to Az, since 100-fold higher drug concentrations are required to obtain an inhibition. With the assumption that T helper cells are likely to be highly sensitive to Az, the effect of low Az concentrations on the other two cell populations involved in T cell-dependent responses has been evaluated. Adherent cells appear unaffected. In contrast, the B-cell response is markedly sensitive to Az, as shown by the effect of Az on the response of nude mouse cells to a T cell-dependent antigen in the presence of T-cell products, either specific or non-specific. On the other hand, the B-cell response to mitogens is resistant to az. Thus, Az has a differential effect on B-cell response according to the thymus dependency of the antigen. This may suggest the existence of two pathways for B-cell activation or two different B-cell subpopulations.

The role of macrophages in the in vitro response of mouse spleen cells to the insolubilized, T-independent antigen trinitrophenylated polyacrylamide (TNP-PAA) is demonstrated by the following points. The response is abolished by filtration on Sephadex G-10 and can be restored by the addition of splenic adherent cells, deficient in either B or T cells, or by 2-mercaptoethanol (2ME). It is suppressed upon elimination of phagocytic cells by silica, and restored by 2-ME. 2-ME can restore a normal response from zero, in cultures depleted of both adherent and phagocytic cells, and is efficient in the absence of mature T cells. Experiments in microcultures show that large numbers of macrophages can stimulate a supra-optimal response from B cells. This response is only obtained in the presence of the antigen, and is specific for TNP. These results show that microphages, probably by their polyclonal B-cell activator (PBA) property, play a role in the specific response to TNP-PAA. This prompts us to discuss the respective roles in the B-cell response of this PBA activity and of the interaction of the antigen with the specific B-cell receptors.

A specific IgM antibody response toward the trinitrophenol (TNP) hapten can be induced in mononuclear blood cell suspensions upon culture with a particulate antigen: polyacrylamide beads conjugated with the TNP hapten (TNP-PAA). The response, and its specificity, are demonstrated by an increase in the number of TNP binding B lymphocytes (specific rosette forming cells), by the appearance of cells producing anti-TNP antibody at a high rate (haemolytic plaques), (ELISA test). The anti-TNP response requires monocytes, the role of which is to produce interleukin-1 (IL-1) and T lymphocytes (belonging to the T4 helper subset) the role of which is to produce interleukins (the characterization of which is under study). We propose a model or B cell activation based on the following signals: an early specific signal, provided by the particulate antigen; several non specific signals, provided by T derived interleukins. The anti-TNP response is negatively regulated by monocytes, the functional states of which can be modified in certain situations (autoimmunity, aging) or influenced by glucocorticoids. Suppressor T lymphocytes of this response (not exclusively of the T8 phenotype) can be induced and this can allow the evaluation of T suppressor cell function. This was used in adult idiopathic thrombocytopenic purpura treated with high doses of intra-venous gammaglobulins.

We tested the effect of interleukin (IL) 4 on the specific IgM antibody response induced by trinitrophenylated-polyacrylamide beads (TNP-PAA) in cultures of human B cells. T cell help was provided by exogeneous IL2. IL4 profoundly suppressed the response to optimal concentrations (50 U/ml) of IL2, with a 50% inhibitory concentration of 6 U/ml. This was due neither to a shift in the kinetics nor to a switch to an IgG response. The production of anti-TNP antibody (as measured by an enzyme-linked immunosorbent assay in the culture supernatant) was inhibited to the same extent as the generation of plaque-forming cells. The effect of IL4 was completely abolished by a neutralizing antibody toward IL4. Kinetic studies showed that IL4 had to be present during the first 48 h of culture to fully inhibit the response. The sequential stimulation of B cells by antigen and by IL2 showed that IL4 does not negatively interfere with signaling through membrane Ig but counteracts the effect of IL2 on antigen-activated B cells.

New methods allow the study of humoral immunity in man using blood lymphocyte cultures. A primary in vitro antibody response can be induced, characterized by the appearance of antibody forming cells, and its pattern is as follows: stringent culture requirements, which have been so far obtained in few laboratories; the kinetics of the response, with a peak on day 7--8; and exclusively IgM response; a T-cell requirement. Among these methods, the model we have described has the advantage of allowing a reproductible response in a given individual. The in vitro study of humoral immunity has limitations, the most apparent of which is that it addresses itself to only one lymphoid population, that in peripheral blood, easily accessible. It presents several advantages, as compared to an in vivo study after injection of an antigen: absence of ethical problem; the necessity of only one blood sample, at a given time, avoiding the interference of a treatment started after the test; the possibility of performing successive primary stimulations for a longitudinal study.

In order to analyze the effects of interleukin (IL) 6 on human in vitro Ig production B lymphocytes were activated by Staphylococcus aureus Cowan strain I (SAC) in the presence of low concentrations of IL2 (1 U/ml) and dexamethasone (10(-7) M). Previously we showed that this model of B cell response is completely monocyte dependent. We here demonstrate that, under these experimental conditions, IL6 is able to replace monocytes and stimulate Ig production provided IL1 is also present. Dose-effect curves show that these two monokines act synergistically. This synergy is demonstrable only in the presence of dexamethasone, when B lymphocytes are activated (by SAC) and when T cell help (provided by IL2) is present. It results in the production of both IgM and IgG. Both IL1 and IL6 have to be present during the first 48 h of culture to exert an optimal effect. These results show that IL6 may act on early (as well as on late) stages of normal B lymphocyte differentiation. Moreover, glucocorticosteroids potentiate the synergistic effect of IL1 and IL6 on their B lymphocyte target, an effect comparable to that exerted on hepatocytes.