Ronni Rômulo Novaes e Brito

B-1 cells are pivotal for in vivo inflammatory giant cell formation

The mechanisms that govern giant cell (GC) formation in inflammatory, neoplastic and physiologic conditions are far from being understood. Here, we demonstrate that B‐1 cells are essential for foreign‐body GC formation in the mouse. GCs were analysed on the surface of glass cover slips implanted into the subcutaneous tissue of the animals. It was demonstrated that GCs are almost absent on cover slips implanted into the subcutaneous tissue of BALB/c or CBA/N X‐linked immunodeficient mice. As these animals do not have B‐1 cells in the peritoneal cavity, they were reconstituted with B‐1 cells obtained from cultures of adherent mouse peritoneal cells. Results showed that in B‐1‐reconstituted animals, the number of GCs on the implant surface surpassed the values obtained with preparations from wild animals. In animals selectively irradiated (pleural and peritoneal cavities) to deplete these cavities of B‐1 cells, GCs were also not formed. Enriched suspensions of B‐1 cells grown in culture were labelled with [3H]‐tymidine and injected into the peritoneal cavity of naive mice before implantation of glass cover slips. After 4 days, about 17% of mononuclear cells had their nuclei labelled, and almost 70% of GCs had one or more of their nuclei labelled when analysed by histoautoradiographic technique. A few GCs expressed an immunoglobulin M when analysed by immunostaining and confocal microscopy. Overall, these data demonstrate that B‐1 cells are pivotal in the mechanisms of foreign‐body GC formation in the mouse.

Role of distinct immune components in the radiation-induced abrogation of systemic lupus erythematosus development in mice

The New Zealand Black × New Zealand White F1 [(NZB/NZW) F1] mouse develops an autoimmune condition resembling aspects of human systemic lupus erythematosus (SLE). We investigated the effects of a novel prophylactic thoraco-abdominal gamma irradiation protocol on the onset and evolution of lupus in these animals. Survival of irradiated mice was higher when compared with nonirradiated mice. Kidney lesions were milder and autoantibody levels were lower in irradiated mice. To identify possible mechanisms involved in the radiation-induced improvement of disease, distinct components of humoral and cellular immune responses were evaluated. Because B-1 cells are known to be involved in various autoimmune diseases, we investigated the participation of these cells in SLE progression. Unexpectedly, B-1 cells were not depleted in (NZB/NZW) F1, even after several rounds of irradiation. No alterations were found in viability and physiology of B-1 cells in SLE animals with the exception of constitutive overexpression of the anti-apoptotic molecule Bcl-2, which may account for the observed radioresistance. Thus, a role for B-1 cells in murine SLE cannot be excluded, since the irradiation protocol did not effectively eliminate these cells. Additionally, we demonstrate a marked delay in the ability of splenocytes to repopulate the spleen after irradiation in (NZB/NZW) F1, in contrast to leucocytes in other cellular compartments. The implications of these findings for the fate of SLE in this model are discussed. Lupus (2007) 16, 947—954.

Cross talk between peritoneal macrophages and B-1 cells in vitro.

B-1 cells constitute a distinct B cell population with unique phenotypic and functional characteristics. They represent the main B cell population found in mouse peritoneal and pleural cavities. The communication between B-1 cells and peritoneal macrophages has been previously studied, and the effect this interaction has on macrophages has been previously described. Using an in vitro co-culture model, herein we demonstrated that peritoneal macrophages were able to increase survival rates and to stimulate proliferation of B-1 cells. IL-6 was also found to be important in B-1 cell survival; recombinant IL-6 increases the percentage of viable B-1 cells in culture. Furthermore, molecules involved in the IL-6 signaling pathway, such as STAT-3 and Bcl-2, were highly expressed in B-1 cells after co-culture with peritoneal macrophages. IL-6-deficient peritoneal macrophages were not able to increase B-1 cell survival, confirming the importance of this cytokine. Altogether, our results indicate a novel mechanism in which peritoneal macrophages are able to regulate the B-1 population via IL-6 secretion.

Crosstalk between B16 melanoma cells and B-1 lymphocytes induces global changes in tumor cell gene expression

The analysis of gene expression patterns in cancers has improved the understanding of the mechanisms underlying the process of metastatic progression. However, the acquisition of invasive behavior in melanoma is poorly understood. In melanoma, components of the immune system can contribute to tumor progression, and inflammatory cells can influence almost all aspects of cancer progression, including metastasis. Recent studies have attributed an important role to B-1 cells, a subset of B lymphocytes, in melanoma progression. In vitro interactions between B16 melanoma cells and B-1 lymphocytes lead to increased B16 cell metastatic potential, but the molecular changes induced by B-1 lymphocytes on B16 cells have not yet been elucidated. In this study, we used a microarray approach to assess the gene expression profile of B16 melanoma cells following contact with B-1 lymphocytes (B16B1). The microarray analysis identified upregulation in genes involved with metastatic progression, such as ctss, ccl5, cxcl2 and stat3. RT-qPCR confirmed this increase in mRNA expression in B16B1 samples. As previous studies have indicated that the ERK1/2 MAPK cascade is activated in melanoma cells following contact with B-1 lymphocytes, RT-qPCR was performed with RNA from melanoma cells before and after contacting B-1 cells and untreated or treated with ERK phosphorylation inhibitors. The results showed that the expression of stat3, ctss and cxcl2 increased in B16B1 but decreased following ERK1/2 MAPK inhibition. Ccl5 gene expression increased after contacting B-1 cells and was maintained at the same level following inhibitor treatment. Stat3 was verified and validated at the protein level by Western blot analysis. STAT3 expression was also significantly increased in B16B1, suggesting that this pathway can also contribute to the increased metastatic phenotype observed in our model. These results indicated that B-1 cells induce important global gene expression changes in B16 melanoma cells. We also evaluated the relationship of some of the genes identified as differentially expressed and the ERK1/2 MAPK cascade. This work may have important implications for understanding the role of B-1 lymphocytes and the ERK/MAPK cascade in the metastatic process.

B16 melanoma cells increase B-1 cell survival, IL-10 production and radioresistance in vitro.

B-1 cells can be differentiated from B-2 cells because they are predominantly located in the peritoneal and pleural cavities and have distinct phenotypic patterns and activation properties. The role of both cell populations in cancer progression is still controversial. Previous studies have indicated that direct contact between B-1 cells and B16 melanoma tumor cells (B16) increases the metastatic potential of the tumor cells. However, cellular changes that are induced in B-1 cells during the interaction between these two cell types have not been evaluated. In the present study, it is hypothesized that B-1 cells are modified after their interaction with tumor cells, leading to both increased cell viability and rate of proliferation. Additionally, soluble factors that were secreted by B16 cells were sufficient to augment B-1 cell viability and to modify the production of IL-10 by B-1 cells. Impressively, after direct or indirect contact with the B16 cells, B-1 cells became resistant to radiation-induced cell death. Thus, future studies that assess the importance of concomitant immunity and other conventional therapies in cancer treatment are needed.

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.

Chronic Sleep Restriction Impairs the Antitumor Immune Response in Mice

Objectives: Sleep regulates immune function reciprocally and can affect the parameters that are directly involved in the immune response. Sleep deprivation is considered to be a stress-causing factor and is associated with impaired immune activity. It causes increased glucocorticoid concentrations by activating the hypothalamic-pituitary-adrenal axis; this can lead to a series of disorders that are associated with the prolonged or increased secretion of these hormones. The aim of this study was to evaluate the effects of sleep restriction (SR) on the development of pulmonary experimental metastasis and the modulation of the tumor immune response. Methods: The SR protocol was accomplished by depriving C57BL/6 male mice of sleep for 18 h/day for 2, 7, 14, and 21 days. The modified multiple-platforms method was used for SR. Results: The results showed that cytotoxic cells (i.e., natural killer [NK] and CD8+ T cells) were reduced in number and regulatory T cells were predominant in the tumor microenvironment. Sleep-restricted mice also exhibited a reduced number of dendritic cells in their lymph nodes, which may have contributed to the ineffective activation of tumor-specific T cells. Peripheral CD4+ and CD8+ T cells were also reduced in the sleep-restricted mice, thus indicating an immunosuppressive status. Conclusions: Sleep deprivation induces failure in the activity of cells that are important to the tumor immune response, both in the tumor microenvironment and on the periphery. This leads to the early onset and increased growth rate of lung metastasis.

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.

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.