Sleep influences the immune response and the rejection process alters sleep pattern: Evidence from a skin allograft model in mice.

INTRODUCTION: Sleep generally regulates immune functions in a supportive manner and can affect parameters that are directly involved in the rejection process. STUDY OBJECTIVES: The first objective was to assess whether sleep deprivation (SD) or sleep restriction (SR) affects the allograft rejection process in mice. The second objective was to investigate whether the rejection process itself modulates the sleep pattern of allografted mice. DESIGN: Adult BALB/c and C57BL/6J male mice were used as the donors and recipients, respectively, except for the syngeneic group (ISOTX), which received skin from mice of the same strain (C57BL/6J). The recipients were randomly assigned to either one of two control groups - TX (allogenic) or ISOTX (syngeneic) - which underwent stereotaxic surgery to enable sleep recording prior to the allograft but were not sleep deprived; one of two paradoxical sleep deprived groups - SDTX and TXSD - which underwent 72h of continuous SD either before or after the allograft respectively, and one of two sleep restricted groups - SRTX and TXSR - which underwent 21h of SD and 3h of sleep for 15days either before or after the allograft respectively. INTERVENTIONS: The skin allograft was inspected daily to determine the survival time, expected as 8.0±0.4days in this transplant model under no treatment. The sleep pattern was controlled throughout the rejection process in the SD and SR groups. Draining lymph nodes, spleen, blood and skin grafts were harvested on the 5th day after transplantation for evaluation of the immune parameters related to allograft rejection. MEASUREMENTS AND RESULTS: In the control groups, we observed a reduction in paradoxical sleep throughout the entire allograft rejection process. Acute and chronic experimental sleep loss in the SD and SR groups produced marked alterations in the immune response. Both SD and SR prolonged allograft survival compared to the non-sleep-deprived group. There were reductions in the following parameters involved in the allograft rejection under sleep loss: CD4+ and CD8+ T cell subpopulations in the peripheral lymph organs and spleen, circulating sIL-2R levels, graft-infiltrating CD4+ T cells and skin allograft global gene expression. CONCLUSIONS: We provide, as far as we are aware, the first evidence in vivo that the immune response can alter the normal sleep pattern, and that sleep loss can conversely affect the immune response related to graft rejection.

B-1 cells produce insulin and abrogate experimental streptozotocin-induced diabetes.

The participation of B-1 cells in a murine model of spontaneous diabetes has been recently reported. Here, we describe the role of B-1 cells in streptozotocin (STZ) induced diabetes in mice. We demonstrated that XID (B-1 cell-deficient) mice are more susceptible to STZ treatment than WT mice, as evidenced by their higher blood glucose level in response to STZ. Unexpectedly, the XID mice that were i.p. transferred with purified B-1 cells, either before or after the STZ treatment, did not develop diabetes. These cell transfers provided long-lasting protection for the XID mice against STZ-induced diabetes, suggesting that B-1 cells play an important role in the experimental diabetes pathobiology. We also showed that B-1 cell culture supernatants were able to regulate the blood glucose level of the diabetic XID mice, and we identified insulin-producing cells when B-1 cells were differentiated in B-1 cell-derived phagocyte in vitro. These findings provide a novel role for B-1 cells in metabolic processes, presenting a new mechanism to explain the pathogenesis of diabetes and a possible therapeutical target.

B-1 cells contribute to susceptibility in experimental infection with Leishmania (Leishmania) chagasi.

The immune response to leishmaniasis is complex, and the result of infection depends on both the genetic composition of the Leishmania species and the immunity of the host. Clinical and experimental evidence suggest that the activation of B cells leads to exacerbation of visceral leishmaniasis. However, the role of B-1 cells (a subtype of B lymphocytes) in the pathogenesis of experimental visceral leishmaniasis has not yet been elucidated. In this study, we investigated the importance of B-1 cells in experimental infection with Leishmania. (L.) chagasi. Our results showed that BALB/XID mice (X-linked immunodeficient mice which are genetically deficient in B-1 cells) infected with L. (L.) chagasi for 45 days had a significant reduction in parasite load in the spleen when compared with control mice. Cytokine analysis showed that the BALB/XID mice had lower amounts of IL-10 in their sera compared with control group. In addition, the transfer of B-1 cells from wild type mice into IL-10KO animals led to an increase in susceptibility to L. (L.) chagasi infection in the IL-10KO mice, suggesting that the IL-10 produced by these cells is important in experimental infection. Our results suggest that B-1 cells may play an important role in susceptibility to L. (L.) chagasi.

Gene expression in B-1 cells from lupus-prone mice.

New Zealand Black X New Zealand White F1 [(NZB/NZW)F1] mice develop an autoimmune condition with similarities to human systemic lupus erythematosus (SLE). In this study, we demonstrate that B-1 cells, which have previously been reported to be involved in several autoimmune diseases, have altered gene expression in these mice. RNA was extracted from purified B-1 cells of disease-free C57BL/6 mice and lupus-prone (NZB/NZW)F1 mice. Gene expression was analysed using DNA microarray techniques and validated by real time reverse transcriptase polymerase chain reaction (RT-PCR). In (NZB/NZW)F1 mice, some genes had altered expression patterns compared to disease-free controls. Specifically, the upregulation of Ifitm1, Pvrl2 and Ifi202b and downregulation of Trp53bp1 mRNA were observed in (NZB/NZW)F1 mice. These genes are known to be associated with autoimmune diseases. This pattern of gene expression in B-1 cells could understanding of the pathogenesis of SLE. Thus, it is reasonable to hypothesise that the altered gene expression observed in B-1 cells in our experimental model is important for SLE prognosis and therapy, and these implications are discussed herein.

Increased bone loss and amount of osteoclasts in kinin B1 receptor knockout mice.

AIM: The pathophysiology of periodontal diseases involves aspects of immunity and bone remodelling. Considering the role of the kinin B1 receptor (Bdkrb1) in inflammation and healing, the purpose of this study was to evaluate the contribution of Bdkrb1 to the pathogenesis of periodontitis. MATERIAL AND METHODS: We used a model of ligature-induced experimental periodontitis (LIEP) in mice lacking Bdkrb1 (Bdkrb1(-/-) ) to test the role of this receptor in bone loss and cytokine secretion by lymph nodes cells. Angiotensin-converting enzyme inhibitor (ACEi) was used as a pharmacological strategy to support the genetic model. Also, autonomous effect of Bdkrb1 deletion was evaluated in osteoclasts precursors from bone marrow. RESULTS: Bdkrb1(-/-) mice exhibit increased bone loss and IL-17 secretion in response to LIEP when compared to wild type. LIEP does not modify TNF-α, IFN-γ and IL-10 levels in Bdkrb1(-/-) mice after 21 days. Bone marrow cells from Bdkrb1(-/-) displayed increased differentiation into functional osteoclasts with consistent artificial calcium phosphate degradation. Furthermore, treatment of mice with ACEi prevented bone destruction. CONCLUSION: Bdkrb1 participates in the pathogenesis of LIEP bone loss possibly through mechanisms that involve modulation of the TH 17 response, thereby demonstrating its role in the development of periodontitis.

Synthetic peptides mimic gp75 from Paracoccidioides brasiliensis in the diagnosis of paracoccidioidomycosis.

Paracoccidioidomycosis (PCM) is a systemic granulomatous disease, endemic in Latin America, caused by the thermal dimorphic fungus Paracoccidioides brasiliensis. Although some fungal antigens have already been characterized and used for serological diagnosis, cross-reactions have been frequently observed. Thus, the examination of fungal forms in clinical specimens or isolation of P. brasiliensis by culture is still the most frequent method for the diagnosis of this mycosis. In this study, a random peptide phage display library was used to select mimotopes of P. brasiliensis, which were employed as antigens in an indirect enzyme-linked immunosorbent assay. The protective monoclonal antibody against experimental PCM (anti-gp75) was used as molecular target to screen a phage display library. That approach led to a synthetic peptide named P2, which was synthesized and tested against PCM patients' sera to check whether it was recognized. There was significant recognition of P2 by sera of untreated PCM patients when compared with normal human sera. Sera from treated PCM group, patients with other mycosis or co-infected with HIV had much lower recognition of P2 than untreated patient group. The test showed a sensitivity of 100 and 94.59% of specificity in relation to human sera control. These data indicate a potential use of P2 as diagnostic tool in PCM. Its application for serological diagnosis of PCM may contribute to the development and standardization of simpler, faster and highly reproducible immunodiagnostic tests at low cost.

Photodynamic therapy for acne vulgaris: a critical review from basics to clinical practice: part I. Acne vulgaris: when and why consider photodynamic therapy?

UNLABELLED: The first-line treatments for acne vulgaris are conventional topical and/or oral medications. However, many patients have contraindications, only partial response, significant adverse effects, or recurrence. Light-based treatments and photodynamic therapy (PDT) using topical precursors of porphyrins are off-label alternative treatments for acne vulgaris, with their own advantages and disadvantages. To date, there is no consensus on PDT methodology and parameters. An understanding of pathophysiology of acne, light-tissue interactions, and PDT mechanisms of action is helpful whenever PDT is considered as an alternative treatment. In general, blue light alone improves acne because of its antiinflammatory effects. PDT using 5-aminolevulenic acid (ALA) or ALA derivatives induces transient antimicrobial and antiinflammatory effects. At high doses, red light PDT may induce inhibition or destruction of sebaceous glands, resulting in clinical improvement. LEARNING OBJECTIVES: After completing this learning activity, participants should be able to compare different treatments of acne, recognize when photodynamic therapy may be a useful off-label treatment for acne vulgaris, and identify variables that may affect the efficacy of photodynamic therapy.

Peptides from Paracoccidioides brasiliensis GP43 inhibit macrophage functions and inflammatory response.

Paracoccidioidomycosis (PCM) is a systemic granulomatous disease caused by Paracoccidioides brasiliensis (Pb), a thermal dimorphic fungus. Its major antigen is a 43-kDa glycoprotein. Gp43 embodies different functions: it participates in evasion mechanisms during the installation of primary infection, stimulates granuloma-like formation in vitro and presents T-cell epitopes that induce protective response against the fungus. Here, we investigated epitopes from gp43 inhibitory of both, macrophage functions and inflammatory reaction. Different gp43 peptides, spanning the entire sequence of the molecule, were added to cultures of bone marrow-derived macrophages. After challenge with zymosan or Pb cells, phagocytic indexes were measured. Peptides expressed on the molecule surface were determined by graphic analysis using the Protean module; DNAstar Inc. Two peptides which decreased phagocytic index and were expressed at the surface of the molecule, P4 and P23, were selected for further studies. It was shown that both inhibited the release of NO by zymosan stimulated macrophages while enhanced release of H(2)O(2). The release of TNF-alpha in culture supernatants from in vitro phagocytic tests showed different response depending of P4 concentration (data not shown). In vivo assays with Mycobacterium bovis - bacillus Calmette-Guérin (BCG) or Pb cells demonstrated that these peptides presented non-specific and specific anti-inflammatory properties.

B-1 cell: the precursor of a novel mononuclear phagocyte with immuno-regulatory properties.

Characterization of the origin, properties, functions and fate of cells is a fundamental task for the understanding of physiological and pathological phenomena. Despite the bulk of knowledge concerning the diverse characteristics of mammalian cells, some of them, such as B-1 cells, are still poorly understood. Here we report the results obtained in our laboratory on these cells in the last 10 years. After showing that B-1 cells could be cultured and amplified in vitro, a series of experiments were performed with these cells. They showed that B1 cells reside mostly in the peritoneal and pleural cavities, migrate to distant inflammatory foci, coalesce to form giant cells and participate in granuloma formation, both in vitro and in vivo. They are also able to present antigens to immunologically responsive cells and are endowed with regulatory properties. Further, we have also shown that these cells facilitate different types of infection as well as tumor growth and spreading. These data are presently reviewed pointing to a pivotal role that these cells may play in innate and acquired immunity.

B-1 lymphocytes increase metastatic behavior of melanoma cells through the extracellular signal-regulated kinase pathway.

Increasing evidence indicates that tumors require a constant influx of myelomonocytic cells to support their malignant behavior. This is caused by tumor-derived factors, which recruit and induce functional differentiation of myelomonocytic cells, most of which are macrophages. Although myeloid lineages are the classical precursors of macrophages, B-lymphoid lineages such as B-1 cells, a subset of B-lymphocytes found predominantly in pleural and peritoneal cavities, are also able to migrate to inflammatory sites and differentiate into mononuclear phagocytes exhibiting macrophage-like phenotypes. Here we examined the interplay of B-1 cells and tumor cells, and checked whether this interaction provides signals to influence melanoma cells metastases. Using in vitro coculture experiments we showed that B16, a murine melanoma cell line, and B-1 cells physically interact. Moreover, interaction of B16 with B-1 cells leads to up-regulation of metastasis-related gene expression (MMP-9 and CXCR-4), increasing its metastatic potential, as revealed by experimental metastases assays in vivo. We also provide evidence that B16 cells exhibit markedly up-regulated phosphorylation of the extracellular signal-regulated kinase (ERK) when cocultured with B-1 cells. Inhibition of ERK phosphorylation induced by B-1 cells with inhibitors of MEK1/2 strongly suppressed the induction of MMP-9 and CXCR-4 mRNA expression and impaired the increased metastatic behavior of B16. In addition, constitutive levels of ERK1/2 phosphorylation in B-1 cells are necessary for their commitment to affect the metastatic potential of B16 cells. Our findings show for the first time that B-1 lymphocytes can contribute to tumor cell properties required for invasiveness during metastatic spread.

B-1 cells facilitate Paracoccidioides brasiliensis infection in mice via IL-10 secretion.

Protective immunity in paracoccidioidomycosis is mainly mediated by cellular immunity. The role of B cells in this disease, in particular B-1 cells, is poorly understood. The aim of this study was to characterize the participation of B-1 cells in resistance or susceptibility of BALB/c and BALB/Xid mice to P. brasiliensis (Pb) pulmonary infection. BALB/Xid, which lacks B-1 cells, exhibited higher resistance to infection when compared with BALB/c mice. However, adoptive transfer of B-1 cells to BALB/Xid mice drastically increased the susceptibility of these animals to Pb infection. The fungal burden in BALB/c and B-1-reconstituted BALB/Xid was significantly higher as compared to BALB/Xid strain. Compact, well-organized granulomas were observed in the lungs of BALB/Xid mice, whereas large lesions with necrotic center with a plethora of fungi developed in BALB/c mice. It was also shown that B-1 cells impair phagocytosis of Pb by macrophages in vitro via secretion of IL-10, which was increased upon stimulation with a purified Pb antigen, gp43. Finally, in vivo blockade of IL-10 led to a better control of infection by the highly susceptible B10.A mouse. These findings suggest that B-1 cells play a major role in resistance/susceptibility to Pb infection in murine models, most likely via production of IL-10.

A cell surface 230 kDa protein from murine melanoma involved with tumor malignancy.

Because melanoma incidence has increased at a dramatic rate, it is relevant to identify novel melanoma antigens for diagnosis and develop monoclonal antibodies recognizing such molecules. Some monoclonal antibodies (mAbs), raised against murine melanoma, identify molecules correlated with carcinogenesis. Herein, we describe a murine melanoma-associated 230 kDa molecule, expressed only in tumorigenic cell lines. Moreover, its expression is higher in more metastatic than less metastatic cells. G12F2 mAb, produced against this antigen, inhibited in vitro proliferation of both murine and human melanoma cells and enhanced in vitro complement activity. It also affected in vivo tumor growth and lung metastases formation. This 230kDa molecule represents an important target for experimental melanoma studies and may become a potential diagnostic marker for malignancy as well as a useful tool for immunotherapeutic approaches.

A surface 75-kDa protein with acid phosphatase activity recognized by monoclonal antibodies that inhibit Paracoccidioides brasiliensis growth.

Paracoccidioides brasiliensis is a thermo-dimorphic fungus responsible for paracoccidioidomycosis (PCM), a systemic granulomatous mycosis prevalent in Latin America. The fungus releases many antigens which may be transiently bound to its cell surface. Some of them may show enzymatic functions essential for maintaining many cell processes and survival of the microorganism at different conditions. In this study, we report the characterization of a secreted 75kDa protein from P. brasiliensis with phosphatase activity. Biologic function of the molecule was demonstrated using two specific mAbs produced and characterized as IgM and IgG isotypes. Confocal microscopy and flow cytometry analysis demonstrated that both mAbs recognized the protein on the fungus surface, mainly in its budding sites. In vitro experiments showed that fungal growth was inhibited by blocking the protein with mAbs. In addition, opsonized yeast cells with both mAbs facilitated phagocytosis by murine peritoneal macrophages. Passive immunization using mAbs before P. brasiliensis mice infection reduced colony-forming units (CFU) in the lungs as compared with controls. Histopathology showed smaller inflammation, absence of yeast cells and no granuloma formation.

Tolerogenic property of B-1b cells in a model of allergic reaction.

Since B-1 cells were first described, their origin and function remain controversial. Given the ability to produce natural antibodies and large amounts of IL-10, there is a consensus about their role in innate immunity. More recently, however, B-1 cells have been associated to adaptive immunity as well, due to the demonstration of immunological memory and antigen presentation capability. Here we demonstrate that adoptive transfer of pre-sensitized B-1b cells (obtained from OVA-sensitized mice) to naïve B-1 deficient animals, drastically affects the ability of transplanted animals to mount an adaptive response upon immunization with OVA. In contrast to naïve B-1 populated mice, mice transplanted with sensitized B-1 exhibit lower anti-OVA antibody levels, milder footpad swelling in response to OVA subcutaneous injection and reduced granulomatous reaction to OVA-coated beads. Moreover, we show that these pre-sensitized B-1 cells, when acting as APCs, induce poor T cell proliferation in vitro when compared with macrophages or B-1 cells obtained from naïve mice. This property may be due in part to insufficient expression of the co-stimulatory molecule CD86, necessary for optimal antigen presentation. In conclusion, our data suggest a novel role for B-1 cells as part of suppressor mechanisms in the immune system.

The axis IL-10/claudin-10 is implicated in the modulation of aggressiveness of melanoma cells by B-1 lymphocytes.

B-1 lymphocytes are known to increase the metastatic potential of B16F10 melanoma cells via the extracellular signal-regulated kinase (ERK) pathway. Since IL-10 is associated with B-1 cells performance, we hypothesized that IL-10 could be implicated in the progression of melanoma. In the present work, we found that the C57BL/6 mice, inoculated with B16F10 cells that were co-cultivated with B-1 lymphocytes from IL-10 knockout mice, developed fewer metastatic nodules than the ones which were injected with the melanoma cells that were cultivated in the presence of wild-type B-1 cells. The impairment of metastatic potential of the B16F10 cells was correlated with low activation of the ERK signaling pathway, supporting the idea that the production of IL-10 by B-1 cells influences the behavior of the tumor. A microarray analysis of the B-1 lymphocytes revealed that IL-10 deficiency is associated with down-regulation of the genes that code for claudin-10, a protein that is involved in cell-to-cell contact and that has been linked to lung adenocarcinoma. In order to determine the impact of claudin-10 in the cross-talk between B-1 lymphocytes and the B16F10 tumor cells, we took advantage of small interfering RNA. The silencing of claudin-10 gene in B-1 lymphocytes inhibited activation of the ERK pathway and abrogated the B-1-induced aggressive behavior of the B16F10 cells. Thus, our findings suggest that the axis IL-10/claudin-10 is a promising target for the development of therapeutic agents against aggressive melanoma.

Granuloma formation in vitro requires B-1 cells and is modulated by Paracoccidioides brasiliensis gp43 antigen.

The mechanisms that determine granuloma formation and the significance of this type of inflammatory response in the pathogenesis of fungal diseases such as paracoccidioidomycosis are far from fully understood. We developed a granuloma model in vitro using beads to evaluate the role of isolated mouse peritoneal macrophages and B-1 cells. We also investigated granuloma formation in the presence of gp43, the main antigenic component of Paracoccidioides brasiliensis, which is secreted exocellularly. To determine whether B-1 cells, macrophages, or both, participate in granuloma formation, peritoneal cells from Xid mice, which lack B-1 cells, were used. Granuloma-like structures were not formed with Xid peritoneal cells or with cells from wild type mice that had their peritoneal and pleural cavities irradiated before the cultures were established. Granulomas were observed either when total adherent peritoneal cells or when isolated B-1 cells were added to macrophage cultures. The data strongly suggest that an interaction of B-1 cells and macrophages plays an important role in granuloma-like formation in this experimental model and that the presence of gp43 strongly stimulates this response.

In vivo and in vitro effect of killed Propionibacterium acnes and its purified soluble polysaccharide on mouse bone marrow stem cells and dendritic cell differentiation.

Among the effects exerted by Propionibacterium acnes, a most relevant one is its capacity to modulate the Th1/Th2 cellular immune response. This effect depends on the induction and activation of antigen presenting cells, mainly dendritic cells (DCs), whose number is increased in the peripheral blood of animals treated with this bacterium. A soluble P. acnes polysaccharide (PS) extract also acts on DCs, modulating a Th1 immune response. These data led us to investigate the role of P. acnes and its soluble PS on murine bone marrow (BM) DCs. Bone marrow cells were analyzed by flow cytometry, showing an increase of stem cells and DCs in P. acnes- or PS-treated animals. Culturing in the presence of granulocyte monocyte colony stimulating factor (GM-CSF) increased the in vitro differentiation and maturation of these cells into BM-derived DCs (CD11c+ and MHC class II+). Maturation of DCs was determined by increased CD80 and CD86 expression, IL-4 and IL-12 production, reduction in phagocytic capacity and increase in the antigen presenting ability to primed or naïve T lymphocytes. These data indicate that P. acnes as well as its PS can modulate BM stem cells, originating mature DCs, which are important mainly at the initial antigen contact.

Down-regulation of dendritic cell activation induced by Paracoccidioides brasiliensis.

Paracoccidioidomycosis (PCM) endemic in Latin America, is a progressive systemic mycosis caused by Paracoccidioides brasiliensis (Pb). The infection can evolve to different clinical forms that are associated to various degrees of suppressed cell-mediated immunity. With the recognition that DCs are able to initiate response in naïve T cells and that they also participate in Th cell education, the present study was undertaken to check whether DCs interact with P. brasiliensis, as well as to elucidate possible mechanisms and consequences of this interaction. Our results indicate that P. brasiliensis infection and purified gp43, its main antigenic component, lead to down-regulation of MHC-II and adhesion properties of immature DCs. The down-regulation was also observed in LPS-induced DC maturation. In addition, an inhibition of IL-12 and TNF-alpha production by both P. brasileinsis or gp43, was observed in LPS-induced DC maturation. These results suggest that protein, released in great amounts by the fungus, might be used, to reduce the effectiveness of the immune response.

Characterization of B-1b cells as antigen presenting cells in the immune response to gp43 from Paracoccidioides brasiliensis in vitro.

Antigen presentation is an essential stage in the development of immune response to a specific antigen. This response can lead to the production of antibodies and/or effector T lymphocyte activation. Macrophages, dendritic cells and B-lymphocytes, among others, act as antigen presenting cells. B-lymphocytes capture antigenic particles through a surface receptor of IgM nature. The interaction IgM-antigen leads to endocytosis of the complex and antigen processing which culminates in presentation of the antigen on the cell surface associated with a class II MHC molecule. At least three B cell subsets, B-1a (Ly-1B), B-1b and B-2, are present in the mouse periphery. B-1a and B1-b cells represent a small population in the adult spleen and are abundant in the peritonial and pleural cavities. It has been demonstrated in our laboratory that B-1b cells spontaneously proliferated in stationary cultures of adherent peritonial cells. Further, that these cells migrate to a non-specific inflammatory focus. Based on these findings, we investigated whether these cells are antigen presenting cells in vitro using as antigenic stimulus gp43 from Paracoccidioides brasiliensis. Results showed that B1-b cells express constitutively high levels of class II MHC and costimulatory molecules inducing an efficient proliferation of gp43 sensitized T lymphocytes.

Modulation of proliferation, differentiation and cytokine secretion of murine B-1b cells by proteins of the extracellular matrix.

At least three B cell subsets, B-1a (Ly-1B), B-1b and B-2, are present in the mouse periphery. B-1a and B1-b cells represent a small population in the adult spleen and are abundant in the peritonial and pleural cavities. It has been demonstrated in our laboratory that B-1b cells spontaneously proliferated in stationary cultures of adherent peritonial cells. Further, that these cells migrate to a non-specific inflammatory focus. Based on these findings, it was investigated whether components of the extracellular matrix (ECM) might selectively influence the adherence, proliferation and cytokine production of these cells in vitro. Results showed that collagen induced a higher level of B-1b cells differentiation into adherent phagocytic cells. It was observed that only fibronectin induced higher level of proliferation than other matrix components. The analysis of cytokine production has shown that the presence of laminin stimulated B-1b cells led to high levels of IL-10 production and fibronectin and collagen induced the production of high levels of TNF-alpha. The combination of fibronectin, collagen and laminin induced higher levels of IL-1beta. These results demonstrate that differentiation, proliferation and cytokine production by B-1b cells are markedly influenced by ECM components.