IL-15 Complexes Induce Migration of Resting Memory CD8 T Cells into Mucosal Tissues.

IL-15 is an essential cytokine known to promote T cell survival and activate the effector function of memory phenotype CD8 T cells. Blocking IL-15 signals also significantly impacts tissue-specific effector and memory CD8 T cell formation. In this study, we demonstrate that IL-15 influences the generation of memory CD8 T cells by first promoting their accumulation into mucosal tissues and second by sustaining expression of Bcl-6 and T-bet. We show that the mechanism for this recruitment is largely dependent on mammalian target of rapamycin and its subsequent inactivation of FoxO1. Last, we show that IL-15 complexes delivered locally to mucosal tissues without reinfection is an effective strategy to enhance establishment of tissue resident memory CD8 T cells within mucosal tissues. This study provides mechanistic insight into how IL-15 controls the generation of memory CD8 T cells and influences their trafficking and ability to take up residence within peripheral tissues.

Rheumatoid arthritis vaccine therapies: perspectives and lessons from therapeutic ligand epitope antigen presentation system vaccines for models of rheumatoid arthritis.

The current status of therapeutic vaccines for autoimmune diseases is reviewed with rheumatoid arthritis as the focus. Therapeutic vaccines for autoimmune diseases must regulate or subdue responses to common self-antigens. Ideally, such a vaccine would initiate an antigen-specific modulation of the T-cell immune response that drives the inflammatory disease. Appropriate animal models and types of T helper cells and signature cytokine responses that drive autoimmune disease are also discussed. Interpretation of these animal models must be done cautiously because the means of initiation, autoantigens, and even the signature cytokine and T helper cell (Th1 or Th17) responses that are involved in the disease may differ significantly from those in humans. We describe ligand epitope antigen presentation system vaccine modulation of T-cell autoimmune responses as a strategy for the design of therapeutic vaccines for rheumatoid arthritis, which may also be effective in other autoimmune conditions.

B cells expressing IFN-γ suppress Treg-cell differentiation and promote autoimmune experimental arthritis.

Clinical efficacy in the treatment of rheumatoid arthritis with anti-CD20 (Rituximab)-mediated B-cell depletion has garnered interest in the mechanisms by which B cells contribute to autoimmunity. We have reported that B-cell depletion in a murine model of proteoglycan-induced arthritis (PGIA) leads to an increase in Treg cells that correlate with decreased autoreactivity. Here, we demonstrate that the increase in Treg cells after B-cell depletion is due to an increase in the differentiation of naïve CD4(+) T cells into Treg cells. Since the development of PGIA is dependent on IFN-γ and B cells are reported to produce IFN-γ, we hypothesized that B-cell-specific IFN-γ plays a role in the development of PGIA. Accordingly, mice with B-cell-specific IFN-γ deficiency were as resistant to the induction of PGIA as mice that were completely IFN-γ deficient. Importantly, despite a normal frequency of IFN-γ-producing CD4(+) T cells, B-cell-specific IFN-γ-deficient mice exhibited a higher percentage of Treg cells compared with that in WT mice. These data indicate that B-cell IFN-γ production inhibits Treg-cell differentiation and exacerbates arthritis. Thus, we have established that IFN-γ, specifically derived from B cells, uniquely contributes to the pathogenesis of autoimmunity through prevention of immunoregulatory mechanisms.

Tissue specific CD4+ T cell priming determines the requirement for interleukin-23 in experimental arthritis.

INTRODUCTION: Rheumatoid arthritis (RA) is a chronic inflammatory disease with striking heterogeneity in (i) clinical presentation, (ii) autoantibody profiles and (iii) responses to treatment suggesting that distinct molecular mechanisms may underlie the disease process. Proteoglycan-induced arthritis (PGIA) is induced by two pathways either by intraperitoneal (i.p.) or subcutaneous (s.c.) exposure to PG. CD4+ T cells primed by the i.p. route are T helper (Th)1 cells expressing interferon gamma (IFN-γ) whereas CD4+ T cells primed by the s.c. route are Th17 cells expressing interleukin (IL)-17. IL-23 is necessary for maintaining the phenotype of Th17 cells; however, IL-23 is inflammatory independent of IL-17. The aim of this study was to determine if PGIA induced by different routes of immunization is dependent on IL-23. METHODS: BALB/c wild type (WT), IL-12p40-/- and IL-23p19-/- littermate mice were immunized with recombinant G1 (rG1) domain of human PG in adjuvant either i.p. or s.c. and development of arthritis monitored. Joint histology was assessed. CD4+ T cell cytokines in spleen, lymph node (LN), and joint were assessed by intracellular staining and cytokine enzyme-linked immunosorbent assay. RNA transcripts for cytokines and transcription factors were examined. RESULTS: PGIA was suppressed in the p40-/- and p19-/- mice immunized by the s.c. route but only inhibited in p40-/- mice by the i.p. route. The joints of s.c. but not i.p. sensitized mice contained a population of CD4+ T cells expressing single positive IFN-γ and IL-17 and double positive IFN-γ/IL-17 which were dependent on IL-23 expression. The IFN-γ and IL-17 response in spleen and inguinal LN was inhibited in p19-/- mice and p40-/- mice after s.c. immunization, whereas in i.p. immunized p19-/- mice, IL-17 but not IFN-γ was reduced. Inguinal LN CD11c+ dendritic cells (DC) from s.c. immunized, but not spleen DC from i.p. immunized mice, produced IL-23, IL-1ß, and IL-6 and activated T cells to produce IL-17. CONCLUSION: IL-23 is necessary for the activity of Th17 after s.c. immunization and does not play a role independent of IL-17 after i.p. immunization. These data demonstrate that the molecular pathways IL-23/17 and IL-12/IFN-γ may represent subtypes of arthritis determined by the mode of induction.

Recruitment of Cbl-b to B cell antigen receptor couples antigen recognition to Toll-like receptor 9 activation in late endosomes.

Casitas B-lineage lymphoma-b (Cbl-b) is a ubiquitin ligase (E3) that modulates signaling by tagging molecules for degradation. It is a complex protein with multiple domains and binding partners that are not involved in ubiquitinating substrates. Herein, we demonstrate that Cbl-b, but not c-Cbl, is recruited to the clustered B cell antigen receptor (BCR) and that Cbl-b is required for entry of endocytosed BCRs into late endosomes. The E3 activity of Cbl-b is not necessary for BCR endocytic trafficking. Rather, the ubiquitin associated (UBA) domain is required. Furthermore, the Cbl-b UBA domain is sufficient to confer the receptor trafficking functions of Cbl-b on c-Cbl. Cbl-b is also required for entry of the Toll-like receptor 9 (TLR9) into late endosomes and for the in vitro activation of TLR9 by BCR-captured ligands. These data indicate that Cbl-b acts as a scaffolding molecule to coordinate the delivery of the BCR and TLR9 into subcellular compartments required for productively delivering BCR-captured ligands to TLR9.

B cell-specific expression of inducible costimulator ligand is necessary for the induction of arthritis in mice.

OBJECTIVE: Inducible costimulator (ICOS)-ICOSL interactions are necessary for activation of Teff cells and follicular helper T (Tfh) cells. ICOSL is expressed on B cells, macrophages, and dendritic cells and can be induced on nonhematopoietic cells. The aim of this study was to determine whether expression of ICOSL on B cells is necessary for the development of proteoglycan (PG)-induced arthritis (PGIA). METHODS: PGIA was initiated by immunizing wild-type and ICOSL-deficient (ICOSL(-/-) ) or B cell-specific ICOSL(-/-) chimeric BALB/c mice with human PG in adjuvant. The onset and severity of arthritis were monitored over time. CD4+ T cell proliferation and CD4+ T cell cytokine production were measured in vitro after the cells were restimulated with PG. Germinal center (GC) B cells, plasma cells, Tfh cells, and Treg cells were identified by staining with specific antibodies. RESULTS: Arthritis progression was completely inhibited in both ICOSL(-/-) mice and B cell-specific ICOSL(-/-) chimeric mice. Production of the Teff cell-produced cytokines interferon-γ and interleukin-17 (IL-17) and the antiinflammatory cytokine IL-4 was suppressed. The reduced percentages of GCs and Tfh cells and the decreased production of IL-21 correlated with a decrease in the anti-mouse PG antibody response. However, the percentage of plasma cells was not reduced despite a reduction in IgG responses. CONCLUSION: These data indicate that the signals provided by ICOSL-expressing B cells to Teff cells and Tfh cells are necessary for the development of arthritis. Thus, therapeutic blockade of ICOSL-ICOS interactions may be an effective strategy for the treatment of rheumatoid arthritis.

Location of CD4+ T cell priming regulates the differentiation of Th1 and Th17 cells and their contribution to arthritis.

Th cytokines IFN-γ and IL-17 are linked to the development of autoimmune disease. In models of rheumatoid arthritis, that is, proteoglycan (PG)-induced arthritis, IFN-γ is required, whereas in collagen-induced arthritis, IL-17 is necessary for development of arthritis. In this study we show that the route of immunization determines the requirement for either IFN-γ or IL-17 in arthritis. Intraperitoneal immunization with PG induces a CD4(+) T cell IFN-γ response with little IL-17 in the spleen and peripheral lymph nodes. However, s.c. immunization induces both an IFN-γ and an IL-17 CD4(+) T cell response in spleen and lymph nodes. The failure to induce a CD4(+) T cell IL-17 response after i.p. immunization is associated with T cell priming, as naive T cells activated in vitro were fully capable of producing IL-17. Moreover, PG-induced arthritis is converted from an IFN-γ to an IL-17-mediated disease by altering the route of immunization from i.p. to s.c. The histological appearance of joint inflammation (cellular inflammation and bone erosion) is similar in the i.p. versus s.c. immunized mice despite the presence of CD4(+) T cells producing IL-17 in joint tissues only after s.c. immunization. These data indicate a critical role for the site of initial T cell priming and the Th cytokines required for susceptibility to arthritis. Our findings suggest that T cell activation at different anatomical sites in rheumatoid arthritis patients may skew the T cells toward production of either IFN-γ or IL-17.

Synovial fluid from patients with early osteoarthritis modulates fibroblast-like synoviocyte responses to toll-like receptor 4 and toll-like receptor 2 ligands via soluble CD14.

OBJECTIVE: Synovial inflammation, a feature of both osteoarthritis (OA) and meniscal injury, is hypothesized to be triggered in part via stimulation of Toll-like receptors (TLRs). We undertook this study to test whether a TLR-2- or TLR-4-stimulating factor in synovial fluid (SF) from patients with early knee OA with meniscal injury could lead to inflammatory activation of synoviocytes. METHODS: SF was obtained from patients with early OA cartilage damage undergoing arthroscopic meniscal procedures. SF was used to stimulate primary cultures of fibroblast-like synoviocytes (FLS) and cell lines transfected with TLR-2 or TLR-4. SF was used either alone or in combination with a TLR-2 stimulus (palmitoyl-3-cysteine-serine-lysine-4 [Pam3CSK4]) or a TLR-4 stimulus (lipopolysaccharide [LPS]). In blocking experiments, SF was preincubated with anti-CD14 antibody. RESULTS: SF from these patients did not stimulate interleukin-8 (IL-8) release from TLR transfectants. Compared with SF on its own, SF (at concentrations of 0.09-25%) in combination with TLR-2 or TLR-4 ligands resulted in significant augmentation of IL-8 release from both transfectants and primary FLS. Soluble CD14 (sCD14), a coreceptor for TLRs, was measured in SF from patients with early OA at levels comparable to those in patients with advanced OA and patients with rheumatoid arthritis. Blockade with anti-CD14 antibody abolished the ability of SF to augment IL-8 production in response to LPS, and diminished Pam3CSK4 responses. CONCLUSION: SF augments FLS responses to TLR-2 and TLR-4 ligands. This effect was largely due to sCD14. Our results demonstrate that sCD14 in the setting of OA and meniscal injury sensitizes FLS to respond to inflammatory stimuli such as TLR ligands.

B effector cells in rheumatoid arthritis and experimental arthritis.

Rheumatoid arthritis is a chronic autoimmune immune disease affecting approximately 1% of the population. There has been a renewed interest in the role of B cells in rheumatoid arthritis based on the evidence that B cell depletion therapy is effective in the treatment of disease. This review summarizes the current knowledge of the mechanisms by which B cells contribute to autoimmune arthritis including roles as autoantibody producing cells, antigen-presenting cells, cytokine producing cells, and regulatory cells.

B cell depletion enhances T regulatory cell activity essential in the suppression of arthritis.

The efficacy of B cell-depletion therapy in rheumatoid arthritis has driven interest in understanding the mechanism. Because the decrease in autoantibodies in rheumatoid arthritis does not necessarily correlate with clinical outcome, other mechanisms may be operative. We previously reported that in proteoglycan-induced arthritis (PGIA), B cell-depletion inhibits autoreactive T cell responses. Recent studies in B cell-depletion therapy also indicate a role for B cells in suppressing regulatory mechanisms. In this study, we demonstrate that B cells inhibited both the expansion and function of T regulatory (Treg) cells in PGIA. Using an anti-CD20 mAb, we depleted B cells from mice with PGIA and assessed the Treg cell population. Compared to control Ab-treated mice, Treg cell percentages were elevated in B cell-depleted mice, with a higher proportion of CD4(+) T cells expressing Foxp3 and CD25. On a per-cell basis, CD4(+)CD25(+) cells from B cell-depleted mice expressed increased amounts of Foxp3 and were significantly more suppressive than those from control Ab-treated mice. The depletion of Treg cells with an anti-CD25 mAb concurrent with B cell-depletion therapy restored the severity of PGIA to levels equal to untreated mice. Although titers of autoantibodies did not recover to untreated levels, CD4(+) T cell recall responses to the immunizing Ag returned as measured by T cell proliferation and cytokine production. Thus, B cells have the capacity to regulate inflammatory responses by enhancing effector T cells along with suppressing Treg cells.

Serum free light chains as biomarkers for systemic lupus erythematosus disease activity.

OBJECTIVE: To evaluate serum free light chains (FLC) as a putative biomarker of systemic lupus erythematosus (SLE) activity. METHODS: Seventy-five SLE patients and 41 age- and sex-matched rheumatoid arthritis (RA) controls were enrolled. Disease activity was assessed using the Safety of Estrogens in Lupus Erythematosus: National Assessment version of the Systemic Lupus Erythematosus Disease Activity Index (SLEDAI) definition and physician global assessments for SLE and the Disease Activity Score in 28 joints for RA. Serum FLC levels were compared against other biomarkers (IgG, C3, C4, double-stranded DNA [dsDNA] antibody). Nonparametric tests were used to compare 1) FLC and IgG in SLE versus RA and healthy controls, 2) FLC and IgG among different levels of activity in SLE, and 3) FLC in active versus nonactive RA. Correlation of FLC, C3, C4, dsDNA antibody, and IgG with the SLEDAI and modified SLEDAI (M-SLEDAI) were obtained. RESULTS: FLC was higher in SLE than in RA; both were higher than referent healthy controls. Total FLC was significantly higher in subjects with greater SLE disease activity than lower/no activity. There were no significant differences in IgG, C4, or dsDNA antibody stratified by disease activity. Total FLC and C3 showed moderate to strong correlation with the SLEDAI and M-SLEDAI. In RA, no differences were seen in FLC levels for different levels of disease activity. Similar results were seen after controlling for renal function, age, and sex. In multiple linear regression, FLC significantly explained 50% variance of the SLEDAI after adjusting for renal function, age, and sex. CONCLUSION: Serum FLC levels correlate strongly with disease activity in SLE, but not in RA. Serum FLC may be used as a biomarker of SLE disease activity.

Proteoglycan-induced arthritis and recombinant human proteoglycan aggrecan G1 domain-induced arthritis in BALB/c mice resembling two subtypes of rheumatoid arthritis.

OBJECTIVE: To develop a simplified and relatively inexpensive version of cartilage proteoglycan-induced arthritis (PGIA), an autoimmunity model of rheumatoid arthritis (RA), and to evaluate the extent to which this new model replicates the disease parameters of PGIA and RA. METHODS: Recombinant human G1 domain of human cartilage PG containing "arthritogenic" T cell epitopes was generated in a mammalian expression system and used for immunization of BALB/c mice. The development and progression of arthritis in recombinant human PG G1-immunized mice (designated recombinant human PG G1-induced arthritis [GIA]) was monitored, and disease parameters were compared with those in the parent PGIA model. RESULTS: GIA strongly resembled PGIA, although the clinical symptoms and immune responses in mice with GIA were more uniform than in those with PGIA. Mice with GIA showed evidence of stronger Th1 and Th17 polarization than those with PGIA, and anti-mouse PG autoantibodies were produced in different isotype ratios in the 2 models. Rheumatoid factor (RF) and anti-cyclic citrullinated peptide (anti-CCP) antibodies were detected in both models; however, serum levels of IgG-RF and anti-CCP antibodies were different in GIA and PGIA, and both parameters correlated better with disease severity in GIA than in PGIA. CONCLUSION: GIA is a novel model of seropositive RA that exhibits all of the characteristics of PGIA. Although the clinical phenotypes are similar, GIA and PGIA are characterized by different autoantibody profiles, and the 2 models may represent 2 subtypes of seropositive RA, in which more than 1 type of autoantibody can be used to monitor disease severity and response to treatment.

B7-H1 expression on non-B and non-T cells promotes distinct effects on T- and B-cell responses in autoimmune arthritis.

The immune system has developed several regulatory mechanisms to maintain homeostasis of adaptive immune responses. T-cell programmed death (PD)-1 recognition of B7-H1 (PD-L1) expressed on APC and non-lymphoid tissue regulates T-cell activation. We show that B7-H1(-/-) mice exhibit exacerbated proteoglycan (PG)-induced arthritis and increased Th-1 CD4(+) T-cell responses. Unexpectedly, the PG-specific antibody response in B7-H1(-/-) mice was diminished. A reduction in the number of peanut agglutinin(+) GC coincided with a decrease in CD19(+) GL-7(+) CD95(+) GC B cells that was a result of increased caspase-induced apoptosis. The percent of CD38(+) CD138(+) emerging plasma cells was decreased. B7-H1(-/-) mice exhibited an increased frequency of CD4(+) PD-1(hi) CXCR5(hi) ICOS(hi) CD62L(lo) T follicular helper cells that displayed a hyperactive phenotype with increased expression of mRNA transcripts for Bcl6, IL-21, and the apoptosis-inducer molecule FasL. In cell transfer of B7-H1(-/-) cells into SCID mice, non-B and non-T cells were sufficient to normalize the antibody response, T-cell hyperactivity, and the development of PG-induced arthritis. These findings indicate that B7-H1 on non-B and non-T cells signals through PD-1 on T effector cells to prevent excessive activation and reduce autoimmune arthritis. Furthermore, these findings demonstrate a novel role for B7-H1 expression in promoting B-cell survival by regulating the activation of T follicular helper cell.

IFN-gamma regulates the requirement for IL-17 in proteoglycan-induced arthritis.

The contribution of the proinflammatory cytokines IFN-gamma and IL-17 to the pathogenesis of experimental arthritis is controversial. In proteoglycan (PG)-induced arthritis (PGIA), severe arthritis is dependent on the production of IFN-gamma, whereas IL-17 is dispensable. In collagen-induced arthritis and Ag-induced arthritis, although high levels of IFN-gamma are secreted, disease is exacerbated in IFN-gamma or IFN-gamma receptor-deficient mice due to the ability of IFN-gamma to suppress IL-17 expression. In the current study, we investigated the effect of IFN-gamma on the IL-17 response and its consequences in PGIA. In PG-immunized IFN-gamma(-/-) mice, despite reduction in arthritis, the PG-specific CD4(+) T cell IL-17 response was significantly increased. Elevated IL-17 contributed to development of arthritis, as disease in IFN-gamma/IL-17(-/-) was significantly reduced in comparison with either IFN-gamma(-/-) or IL-17(-/-) mice. A contribution of IFN-gamma and IL-17 to the development of arthritis was also identified in T-bet(-/-) mice. PG-specific CD4(+) T cells from T-bet(-/-) mice produced reduced IFN-gamma and elevated concentrations of IL-17. Both IFN-gamma and IL-17 contribute to arthritis, as T-bet(-/-) mice lacking IL-17 (T-bet/IL-17(-/-)) were resistant, whereas wild-type, T-bet(-/-), and IL-17(-/-) mice were susceptible to PGIA. T cell proliferation and autoantibody production did not correlate with development of disease; however, expression of cytokines and chemokines in joint tissues demonstrate that IFN-gamma and IL-17 cooperatively contribute to inflammation. These results demonstrate that both IFN-gamma and IL-17 have the potential to induce PGIA, but it is the strength of the IFN-gamma response that regulates the contribution of each of these Th effector cytokines to disease.

HSV type 2 infection increases HIV DNA detection in vaginal tissue of mice expressing human CD4 and CCR5.

The goal of this study was to develop an in vivo murine model that can be used to study the influence of HSV-2 on HIV infection. Mice expressing transgenes for human CD4, CCR5, and Cyclin T1 were infected intravaginally with HSV-2 and 3-7 days later infected with HIV. HIV DNA was detected by real-time PCR. The frequency of detection of HIV DNA was significantly higher (65%) in vaginal tissue of HSV-2-infected mice compared to mock-infected mice (35%) when HIV was given 3 days after HSV-2. HSV-2-infected mice also had significantly higher levels of HIV DNA in vaginal tissue. HIV DNA was not detected in vaginal tissue of mice lacking human CD4. Longer periods (5 or 7 days) between infection with HSV-2 and HIV did not increase the frequency of detection or the amount of HIV DNA detected. HIV DNA was also detected in lymph nodes from some of the mice that were infected intravaginally with HSV-2 and HIV. Flow cytometric and mRNA analysis of human CD4 in vaginal tissue suggested that HSV-2 infection increased the number of T cells expressing human CD4 in vaginal tissue. This study provides evidence that HIV infection of cells occurs in the vagina of mice expressing human CD4, CCR5, and Cyclin T1 and that HSV-2 infection increases HIV infection. These findings demonstrate that this model can be used to study the mechanisms responsible for increased susceptibility to HIV in HSV-2-infected persons and for testing preventative treatments.

CCR5 is involved in resolution of inflammation in proteoglycan-induced arthritis.

OBJECTIVE: CCR5 and its ligands (CCL3, CCL4, and CCL5) may play a role in inflammatory cell recruitment into the joint. However, it was recently reported that CCR5 on T cells and neutrophils acts as a decoy receptor for CCL3 and CCL5 to assist in the resolution of inflammation. The aim of this study was to determine whether CCR5 functions as a proinflammatory or antiinflammatory mediator in arthritis, by examining the role of CCR5 in proteoglycan (PG)-induced arthritis (PGIA). METHODS: Arthritis was induced by immunizing wild-type (WT) and CCR5-deficient (CCR5(-/-)) BALB/c mice with human PG in adjuvant. The onset and severity of PGIA were monitored over time. Met-RANTES was used to block CCR5 in vivo. Arthritis was transferred to SCID mice, using spleen cells from arthritic WT and CCR5(-/-) mice. The expression of cytokines and chemokines was measured by enzyme-linked immunosorbent assay. RESULTS: In CCR5(-/-) mice and WT mice treated with the CCR5 inhibitor Met-RANTES, exacerbated arthritis developed late in the disease course. The increase in arthritis severity in CCR5(-/-) mice correlated with elevated serum levels of CCL5. However, exacerbated arthritis was not intrinsic to the CCR5(-/-) lymphoid cells, because the arthritis that developed in SCID mouse recipients was similar to that in WT and CCR5(-/-) mice. CCR5 expression in the SCID mouse was sufficient to clear CCL5, because serum levels of CCL5 were the same in SCID mouse recipients receiving cells from either WT or CCR5(-/-) mice. CONCLUSION: These data demonstrate that CCR5 is a key player in controlling the resolution of inflammation in experimental arthritis.

Endocytic sequestration of the B cell antigen receptor and toll-like receptor 9 in anergic cells.

In autoimmune prone murine strains, sequential engagement of the B cell antigen receptor (BCR) on the cell surface and toll-like receptors (TLRs) in late endosomes is necessary and sufficient for secretion of autoantibodies. However, ubiquitous nucleoprotein self-antigens fail to elicit productive TLR activation, and break self-tolerance in anergic DNA-reactive B cells. The mechanisms limiting TLR activation in these cells are largely unknown. Here, we demonstrate that in anergic 3H9/Vkappa8 and Ars/A1 B cells the normal endocytic transit of both the ligated BCR and TLR9 into late endosomes is abrogated. The BCR and TLR9 arrest together just outside late endosomes, indicating that they enter this compartment along a single, regulated endocytic route. Access to late endosomes could be restored by reversing anergy through several methods, including conferring genetic susceptibility to autoimmunity, complementing proximal BCR signaling or by preventing BCR binding to self-antigen. Downstream of the BCR, JNK, which is activated in naive but not anergic B cells, regulated entry into late endosomes. Restoration of BCR and TLR9 endocytic trafficking rescued TLR9 activation by BCR-captured ligands. These results indicate that B cell anergy is reinforced by the exclusion of both TLRs and their BCR captured ligands from subcellular environments necessary for TLR activation.

BALB/c mice genetically susceptible to proteoglycan-induced arthritis and spondylitis show colony-dependent differences in disease penetrance.

INTRODUCTION: The major histocompatibility complex (H-2d) and non-major histocompatibility complex genetic backgrounds make the BALB/c strain highly susceptible to inflammatory arthritis and spondylitis. Although different BALB/c colonies develop proteoglycan-induced arthritis and proteoglycan-induced spondylitis in response to immunization with human cartilage proteoglycan, they show significant differences in disease penetrance despite being maintained by the same vendor at either the same or a different location. METHODS: BALB/c female mice (24 to 26 weeks old after 4 weeks of acclimatization) were immunized with a suboptimal dose of cartilage proteoglycan to explore even minute differences among 11 subcolonies purchased from five different vendors. In vitro-measured T-cell responses, and serum cytokines and (auto)antibodies were correlated with arthritis (and spondylitis) phenotypic scores. cDNA microarrays were also performed using spleen cells of naïve and immunized BALB/cJ and BALB/cByJ mice (both colonies from The Jackson Laboratory, Bar Harbor, ME, USA), which represent the two major BALB/c sublines. RESULTS: The 11 BALB/c colonies could be separated into high (n = 3), average (n = 6), and low (n = 2) responder groups based upon their arthritis scores. While the clinical phenotypes showed significant differences, only a few immune parameters correlated with clinical or histopathological abnormalities, and seemingly none of them affected differences found in altered clinical phenotypes (onset time, severity or incidence of arthritis, or severity and progression of spondylitis). Affymetrix assay (Affymetrix, Santa Clara, CA, USA) explored 77 differentially expressed genes (at a significant level, P < 0.05) between The Jackson Laboratory's BALB/cJ (original) and BALB/cByJ (transferred from the National Institutes of Health, Bethesda, MD, USA). Fourteen of the 77 differentially expressed genes had unknown function; 24 of 77 genes showed over twofold differences, and only 8 genes were induced by immunization, some in both colonies. CONCLUSIONS: Using different subcolonies of the BALB/c strain, we can detect significant differences in arthritis phenotypes, single-nucleotide polymorphisms (SNPs), and a large number of differentially expressed genes, even in non-immunized animals. A number of the known genes (and SNPs) are associated with immune responses and/or arthritis in this genetically arthritis-prone murine strain, and a number of genes of as-yet-unknown function may affect or modify clinical phenotypes of arthritis and/or spondylitis.

CD44-specific antibody treatment and CD44 deficiency exert distinct effects on leukocyte recruitment in experimental arthritis.

CD44, the leukocyte adhesion receptor for hyaluronan, has been considered a therapeutic target on the basis of the robust anti-inflammatory effect of CD44-specific antibodies in animal models of immune-mediated diseases. However, CD44 deficiency does not provide substantial protection against inflammation. Using intravital video microscopy in a murine model of rheumatoid arthritis, we show that CD44 deficiency and anti-CD44 antibody treatment exert disparate effects on leukocyte recruitment in inflamed joints. Leukocyte rolling, which is increased in CD44-deficient mice, is promptly abrogated in anti-CD44-treated wild-type mice. CD44-specific antibodies also trigger platelet deposition on granulocytes and subsequent depletion of this leukocyte subset in the circulation. These in vivo effects require CD44 cross-linking and are reproducible with an antibody against Gr-1, a molecule that, like CD44, is highly expressed on granulocytes. Anticoagulant pretreatment, which prevents platelet deposition, mitigates both granulocyte depletion and the suppressive effect of CD44-specific antibody on joint swelling. Our observations suggest that cross-linking of prominent cell surface molecules, such as CD44 or Gr-1, can initiate a rapid self-elimination program in granulocytes through engagement of the coagulation system. We conclude that the robust anti-inflammatory effect of CD44-specific antibodies in arthritis is primarily the result of their ability to trigger granulocyte depletion.