Splenic CD11b Research Articles

NOD2 Transgenic Mice Exhibit Enhanced MDP-Mediated Down-Regulation of TLR2 Responses and Resistance to Colitis Induction

Mutations in the CARD15 gene encoding NOD2 are susceptibility factors in Crohn's disease. We explored the mechanism of this susceptibility using mice that over express NOD2. Cellular and molecular responses of mice bearing an NOD2 transgene or administered plasmids that express wild-type and mutated NOD2 constructs were examined. In initial studies, we showed that splenocytes from NOD2 transgenic mice as compared with littermate controls exhibit decreased interleukin (IL)-12p70 responses to peptidoglycan (PGN), a TLR2 ligand that contains muramyl dipeptide, but not other TLR ligands; in contrast, IL-12 responses to PAM(3)CSK(4), a TLR2 ligand that does not contain muramyl dipeptide, were normal. Similarly, transgenic mice as compared with controls exhibited greatly decreased IL-12p40 responses to intraperitoneal administration of PGN but not to lipopolysaccharide. In further studies, we showed using electrophoretic mobility shift assay that PGN-stimulated cells from transgenic mice exhibited decreased activation of nuclear factor kappaB. Finally, in a series of studies on the effect of the NOD2 on susceptibility to induced colitis, we found that (1) transgenic mice were highly resistant to induction of PGN colitis and partially resistant to induction of trinitrobenzene sulfonic acid (TNBS) colitis and (2) mice administered a plasmid expressing a wild-type NOD2 gene were completely resistant to TNBS colitis whereas mice administered a plasmid expressing an NOD2 gene with the Crohn's disease frameshift mutation were only slightly resistant to TNBS colitis. These data offer new evidence that NOD2 mutations contribute to inflammatory bowel disease by causing excessive TLR2 cytokine responses.

Pathogenesis ofYersinia pestisInfection in BALB/c Mice: Effects on Host Macrophages and Neutrophils

The pathogenesis of infection with Yersinia pestis, the causative agent of plague, was examined following subcutaneous infection of BALB/c mice with a fully virulent strain expressing green fluorescent protein. Plate culturing, flow cytometry, and laser confocal microscopy of spleen homogenates throughout infection revealed three discernible stages of infection. The early phase was characterized by the presence of a small number of intracellular bacteria mostly within CD11b+ macrophages and Ly-6G+ neutrophils. These bacteria were not viable, as determined by plate culturing of spleen homogenates, until day 2 postinfection. Between days 2 and 4 postinfection, a plateau phase was observed, with bacterial burdens of 10(3) to 10(4) CFU per spleen. Flow cytometric analysis revealed that there was even distribution of Y. pestis within both CD11b+ macrophage and Ly-6G+ neutrophil populations on day 2 postinfection. However, from day 3 postinfection onward, intracellular bacteria were observed exclusively within splenic CD11b+ macrophages. The late phase of infection, between days 4 and 5 postinfection, was characterized by a rapid increase in bacterial numbers, as well as escape of bacteria into the extracellular compartment. Annexin V staining of spleens indicated that a large proportion of splenic neutrophils underwent rapid apoptosis on days 1 and 2 postinfection. Fewer macrophages underwent apoptosis during the same period. Our data suggest that during the early stages of Y. pestis infection, splenic neutrophils are responsible for limiting the growth of Y. pestis and that splenic macrophages provide safe intracellular shelters within which Y. pestis is able to grow and escape during the later stages of infection. This macrophage compliance can be overcome in vitro by stimulation with a combination of gamma interferon and tumor necrosis factor alpha.

Tissue-specific alterations in the glucocorticoid sensitivity of immune cells following repeated social defeat in mice

Endogeneous glucocorticoids (GC) play an important role in the termination of the inflammatory response following infection and tissue injury. However, recent findings indicate that stress can impair the anti-inflammatory capacities of these hormones. Lipopolysaccharide (LPS)-stimulated splenocytes of mice that were repeatedly subjected to social disruption (SDR) stress were less sensitive to the immunosuppressive effects of corticosterone (CORT) as demonstrated by an increased production of pro-inflammatory cytokines and enhanced cell survival. Myeloid cells expressing the marker CD11b were shown to play a key role in this process. Here we investigated the role of the bone marrow as a potential source of the GC-insensitive cells. The study revealed that LPS-stimulated bone marrow cells, in the absence of experimental stress, were virtually GC-resistant and retained high levels of cell viability after treatment with CORT. Recurrent exposure to the acute stressor over a period of 2, 4 or 6 days led to an increase in the GC sensitivity of the bone marrow cells. This increase in GC sensitivity was associated with enhanced mRNA expression of granulocyte–macrophage colony-stimulating factor (GM-CSF), an increase in the number of myeloid progenitors, and a decrease in the proportion of mature CD11b + cells. The changes in the cellular composition of the bone marrow were accompanied by an increase in splenic CD11b + cell numbers. Simultaneous assessment of the GC sensitivity in bone marrow and spleen revealed a significant negative correlation between both tissues suggesting that social stress causes the redistribution of GC-insensitive myeloid cells from the bone marrow to the spleen.

Cyclophosphamide Induced CD11b+ Cells Can Inhibit Anti-CD40 Monoclonal Antibody (mAb) Therapy of B-Cell Lymphoma.

Immunotherapy with mAb is emerging as an important component of treatment for B-cell lymphoma. Furthermore, anti-tumor effects may be enhanced by combining mAb with a range of conventional cancer treatments and recently impressive results have been acheived with mAb in combination with chemotherapy. Here, we have assessed the efficacy of combining an agonistic anti-CD40 mAb with cyclophosphamide (CY). We have previously shown that large repeated doses of anti-CD40 mAb alone may stimulate cytotoxic T-cell lymphocyte (CTL) responses and provide robust protection in syngeneic murine tumors, such as BCL1 lymphoma. In this study we therefore used single smaller doses of the mAb that alone fail to provide long-term clearance thus allowing us to look for additive or synergistic effects of anti-CD40 and CY. Interestingly, the timing of CY relative to mAb administration appeared critical to therapeutic outcome. Cohorts given CY 7 days (d7 CY) prior to anti-CD40 show reduced survival compared to those receiving mAb alone. In contrast treatment with CY given 1 day before, or on the same day as mAb resulted in greatly increased tumor free survival. In vivo tracking experiments revealed that there is a potent CTL response that clears tumor around 5 days after anti-CD40 treatment. However, when CY was given d7 prior to anti-CD40 mAb, CTL expansion was reduced compared with other treatments, and instead resulted in increased numbers of splenic CD11b+ cells. These CD11b+ cells have an immature myeloid precursor phenotype and were able to produce nitric oxide (NO). Co-culture of CD11b+ cells with murine splenocytes resulted in greatly reduced cytotoxic CTL proliferation, but no apoptosis, which was reversed in the presence of NO inhibitors. Similarly, splenic CTL proliferation in vivo is inhibited by this increased population of CD11b+ cells. Importantly adoptive transfer of CY-induced CD11b+ into non-CY-treated mice inhibited anti-CD40 mAb tumor protection in BCL1 lymphoma. This data indicate that combining CY with immunotherapy can enhance therapeutic responses, but the timing of CY relative to immunotherapy is critical to the therapeutic outcome.

Interferon‐α mRNA in Splenic CD11b+ Marginal Zone Macrophages of C4‐Deficient Mice

The absence of early complement components (C1, C4 and C2 but not C3) is a predisposing factor for systemic lupus erythematosus (SLE). Recently, we demonstrated that, in C4‐deficient (C4 def.) mice, IgM‐containing immune complexes (IgM‐IC) are filtered by the splenic barrier of marginal zone macrophages (MZM), resulting in an increased immune response against antigens within these IgM‐IC, but this could not be observed in wildtype or C3 def. mice. We hypothesized that splenic CD11b+ MZM play an important role in the induction of autoimmunity, and we therefore analysed their cytokine profile after isolation with the help of magnetic antibody cell sorting. mRNA was isolated, and real‐time PCR was performed with specific primers for murine IFN‐γ (IFN‐γ), interleukin‐12 (IL‐12) and IFN‐α (IFN‐α). We observe a moderate increase of IL‐12 and IFN‐γ mRNA in CD11b+ cells of C4 def. mice compared to wildtype cells. Surprisingly, the concentration of IFN‐α mRNA is six times higher in C4 def. mice. Preliminary results suggest that mRNA in CD11b+ cells of C3 def. mice is even lower than that in wt. Six hours following i.v. application of 20 µg of a murine monoclonal IgM anti‐dsDNA antibody, production of IL‐12, IFN‐γ and IFN‐α mRNA is increased in CD11b+ cells of both C4 def. and wt mice. Several references described increased levels of INF‐α in patients with SLE. Dendritic cells are discussed as a major source of IFN‐α. Our observation that C4‐deficient, SLE‐susceptible mice demonstrate an increased spontaneous IFN‐α production by splenic CD11b+ marginal zone macrophages could be an early sign and a trigger for the development of SLE. This is supported by the fact that the absence of C3 is not a predisposing factor for SLE and our observation that C3 def. animals display low levels of IFN‐α mRNA.

Enhanced osteoclast development in collagen-induced arthritis in interferon-γ receptor knock-out mice as related to increased splenic CD11b+ myelopoiesis

Collagen-induced arthritis (CIA) in mice is accompanied by splenomegaly due to the selective expansion of immature CD11b+ myeloblasts. Both disease manifestations are more pronounced in interferon-γ receptor knock-out (IFN-γR KO) mice. We have taken advantage of this difference to test the hypothesis that the expanding CD11b+ splenic cell population constitutes a source from which osteoclast precursors are recruited to the joint synovia. We found larger numbers of osteoclasts and more severe bone destruction in joints of IFN-γR KO mice than in joints of wild-type mice. Osteoclast-like multinucleated cells appeared in splenocyte cultures established in the presence of macrophage colony-stimulating factor (M-CSF) and stimulated with the osteoclast-differentiating factor receptor activator of NF-κB ligand (RANKL) or with tumour necrosis factor-α (TNF-α). Significantly larger numbers of such cells could be generated from splenocytes of IFN-γR KO mice than from those of wild-type mice. This was not accompanied, as might have been expected, by increased concentrations of the intracellular adaptor protein TRAF6, known to be involved in signalling of RANKL- and TNF-α-induced osteoclast formation. Splenocyte cultures of IFN-γR KO mice also produced more TNF-α and more RANKL than those of wild-type mice. Finally, splenocytes isolated from immunised IFN-γR KO mice contained comparatively low levels of pro-interleukin-1β (pro-IL-1β) and pro-caspase-1, indicating more extensive conversion of pro-IL-1β into secreted active IL-1β. These observations provide evidence that all conditions are fulfilled for the expanding CD11b+ splenocytes to act as a source of osteoclasts and to be indirectly responsible for bone destruction in CIA. They also provide a plausible explanation for the higher susceptibility of IFN-γR KO mice to CIA.

Functional alterations in CD11b+Gr-1+ cells in mice injected with allogeneic tumor cells and treated with 2,3,7,8-tetrachlorodibenzo-p-dioxin

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) exposure results in an increased percentage of CD11b+ (Mac-1+) cells in the spleens of mice challenged with P815 tumor cells, coincident with a failure of the mice to generate allospecific CD8+ CTL activity. Since CD11b+Gr-1+ myeloid suppressor cells (MSC) have been described as that which prevent cytotoxic T lymphocyte (CTL) development in a variety of disease states, we hypothesized that TCDD promoted MSC development, leading to suppression of CTL activity. The purpose of the present studies was to compare the phenotypic and functional characteristics of CD11b+ cells in vehicle- and TCDD-treated mice during the P815 tumor allograft response to determine their potential to function as MSC. Initial studies showed that virtually all splenic CD11b+ cells in both vehicle- and TCDD-treated mice co-expressed Gr-1. Consistent with MSC activity, CD11b+Gr-1+ cells isolated from TCDD- but not vehicle-treated mice suppressed the development of CTL activity when added in vitro to mixed lymphocyte–P815 tumor cell cultures. Also consistent with MSC activity, this suppressive effect in vitro required cell-to-cell contact. Surprisingly, however, in vivo depletion of CD11b+Gr-1+ cells failed to affect TCDD-induced suppression of the CTL response, arguing against an immunoregulatory role for the cells in vivo. Immunohistochemical analysis of the spleen showed that CD11b+Gr-1+ cells were localized in the red pulp, and physically separated from the T cells in the white pulp. The localization of CD11b+Gr-1+ cells in the red pulp was indicative of extramedullary myelopoiesis and suggested that TCDD enhanced myelopoiesis. A significantly enhanced neutrophilia in the blood of TCDD-treated mice supported this conclusion. CD11b+Gr-1+ cells isolated from the blood or spleen of TCDD-treated mice produced up to fivefold higher levels of superoxide following PMA stimulation when compared with cells from vehicle-treated mice. However, unlike vehicle-treated mice, CD11b+Gr-1+ cells from TCDD-treated mice were unable to kill YAC-1 target cells. These results indicate that TCDD exposure alters the host response to allogeneic tumor growth, resulting in enhanced myelopoiesis perhaps as a compensatory response to the suppressed T cell-mediated immunity in the face of an increasing P815 tumor burden. Furthermore, within the context of the P815 response, TCDD appears to alter the functional capabilities of mature neutrophils, by enhancing their oxidative burst capacity but reducing their tumoricidal response.

Parenchymal Microglia of Naı̈ve Adult C57BL/6J Mice Express High Levels of B7.1, B7.2, and MHC Class II

In this study, we addressed B7.1, B7.2, and MHC class II expression on microglia of normal adult C57BL/6J mice, which are susceptible to MOG35-55 peptide-induced experimental autoimmune encephalomyelitis (EAE). We showed that there are two distinct major populations of CD11b+ cells in the central nervous system (CNS) of naı̈ve mice: CD45 low (CD45lo; parenchymal microglia) and CD45 intermediate (CD45int; CNS-associated macrophages). These two populations compose CNS microglia. There is a rare CD45 high (CD45hi) population. By contrast, splenic CD11b+ cells (macrophages) are CD45int and CD45hi, but rarely CD45lo. CD45loCD11b+ cells constitutively express much higher levels of B7.1, B7.2, and MHC class II compared to CD45int CD11b+ cells. A shift of CD11b+ cells from CD45lo to CD45int was observed in the CNS of EAE mice. Our study provides evidence that (1) CD45lo and CD45hi, but not CD45int, could be unique markers to differentiate parenchymal microglia from infiltrating macrophages in EAE; (2) the level of CD45 expression on parenchymal microglia (CD45lo) was upregulated in EAE; and (3) parenchymal microglia in normal CNS could be potent APCs by expressing high levels of B7.1, B7.2, and MHC class II molecules and could therefore play an important role in inflammation and autoimmunity in the CNS.

Characterization of a New Antigen Expressed by B and Myeloid Lineage Cells Identified by the Monoclonal Antibody LIP-6

The LIP-6 MAb was produced against the undifferentiated cell line bh2-1 and recognizes an antigen expressed on all pre-B and B cell lines tested and some myeloid lineage lines. FACS analysis of normal tissues showed that LIP-6 is expressed on B lineage cells at all stages of differentiation, from bone marrow pre-B to plasma cells. T cells and thymocytes are LIP-6−, and splenic CD11b+cells are heterogeneous for LIP-6 expression. The LIP-6 MAb was shown to precipitate a major 75-kDa and a minor 85-kDa protein under reducing conditions and a large protein of >240 kDa under nonreducing conditions. Removal of N-linked sugars from the reduced lysates resulted in a single 65-kDa protein, suggesting that there is differential glycosylation of a single 65-kDa protein that forms disulfide-linked multimers. Finally, the LIP-6 antigen was shown not to be linked to the cell surface via a GPI linkage.