Co-occurrence of non-alcoholic steatohepatitis exacerbates psoriasis associated with decreased adiponectin expression in a murine model.

Introduction: Clinical studies have suggested a bidirectional association between non-alcoholic steatohepatitis (NASH) and psoriasis, affecting each other's development and severity. Here, we explored bidirectional causal linkages between NASH and psoriasis using a murine model. Methods: NASH was induced in mice by streptozotocin injection at 2 days of age and by high-fat diet feeding (STAM™ model). Psoriasis was induced by topical application of imiquimod (IMQ) on the ear. The severities of liver damage and psoriatic skin changes were determined using histological analysis. Gene expression in the skin tissues was evaluated using quantitative PCR analysis. Serum cytokine levels were determined using enzyme-linked immunosorbent assay. To examine the innate immune responses of normal human epidermal keratinocytes (NHEKs), the cells were treated with interleukin (IL)-17A, tumor necrosis factor (TNF)-α, and AdipoRon, an adiponectin receptor agonist. Results and Discussion: There were no differences in the degree of liver tissue damage (fat deposition, inflammation, and fibrosis) between NASH mice with and those without psoriasis. Conversely, the co-occurrence of NASH significantly augmented psoriatic skin changes, represented by epidermal hyperplasia, in psoriatic mice. Pro-inflammatory cytokines were expressed in the inflamed skin of psoriatic mice, and the expression of genes, especially Il23a, Il1b, Il36g, and Mip2, was significantly upregulated by the co-occurrence of NASH. The expression of keratinocyte activation marker genes Defb4b and Krt16 was also upregulated by the co-occurrence of NASH. The serum TNF-α and IL-17 levels were increased by the co-occurrence of NASH and psoriasis. The serum adiponectin levels decreased in NASH mice compared with that in non-NASH mice. In NHEK culture, TNF-α and IL-17A synergistically upregulated CXCL1, CXCL8, and IL1B expression. The upregulated pro-inflammatory gene expression was suppressed by AdipoRon treatment, reflecting the anti-inflammatory capacity of adiponectin. Conclusion: The co-occurrence of NASH exacerbated psoriatic skin changes associated with increased serum inflammatory cytokine levels and decreased serum adiponectin levels. Combined with in vitro findings, increased inflammatory cytokine levels and decreased adiponectin levels likely promote innate immune responses in epidermal keratinocytes in psoriatic skin lesions. Overall, therapeutic intervention for co-occurring NASH is essential to achieve a favorable prognosis of psoriasis in clinical practice.

Imatinib has minimal effects on inflammatory and osteopenic phenotypes in a murine cherubism model.

OBJECTIVE: Cherubism is a genetic disorder characterised by bilateral jawbone deformation. The associated jawbone lesions regress after puberty, whereas severe cases require surgical treatment. Although several drugs have been tested, fundamental treatment strategies for cherubism have not been established. The effectiveness of imatinib has recently been reported; however, its pharmaceutical mechanism remains unclear. In this study, we tested the effects of imatinib using a cherubism mouse model. METHODS: We used Sh3bp2 P416R cherubism mutant mice, which exhibit systemic organ inflammation and osteopenia. The effects of imatinib were determined using primary bone marrow-derived macrophages. Imatinib was administered intraperitoneally to the mice, and serum tumour necrosis factor-α (TNFα), organ inflammation and bone properties were examined. RESULTS: The cherubism mutant macrophages produced higher levels of TNFα in response to lipopolysaccharide compared to wild-type macrophages, and imatinib did not significantly suppress TNFα production. Although imatinib suppressed osteoclast formation in vitro, administering it in vivo did not suppress organ inflammation and osteopenia. CONCLUSION: The in vivo administration of imatinib had a minimal therapeutic impact in cherubism mutant mice. To establish better pharmaceutical interventions, it is necessary to integrate new findings from murine models with clinical data from patients with a definitive diagnosis of cherubism.

TRAPS mutations in Tnfrsf1a decrease the responsiveness to TNFα via reduced cell surface expression of TNFR1.

Tumor necrosis factor (TNF) receptor-associated periodic syndrome (TRAPS) is an autoinflammatory periodic fever syndrome associated with heterozygous mutations in TNFRSF1A, which encodes TNF receptor type I (TNFR1). Although possible proinflammatory mechanisms have been proposed, most previous studies were performed using in vitro overexpression models, which could lead to undesirable inflammatory responses due to artificial overexpression. It is crucial to reproduce heterozygous mutations at physiological expression levels; however, such studies remain limited. In this study, we generated TRAPS mutant mice and analyzed their phenotypes. Three Tnfrsf1a mutant strains were generated by introducing T79M, G87V, or T90I mutation. T79M is a known mutation responsible for TRAPS, whereas G87V is a TRAPS mutation that we have reported, and T90I is a variant of unknown significance. Using these murine models, we investigated whether TRAPS mutations could affect the inflammatory responses in vivo and in vitro. We found that none of the mutant mice exhibited detectable inflammatory phenotypes under standard housing conditions for 1 year. Interestingly, TRAPS mutant (T79M and G87V) mice had reduced mortality rates after the administration of lipopolysaccharide (LPS) and D-galactosamine, which induce TNFα-dependent lethal hepatitis. Moreover, TRAPS mutations strongly suppressed the development of TNFα-mediated arthritis when crossed with human TNFα transgenic mice. In in vitro primary bone marrow-derived macrophage cultures, the T79M and G87V mutations attenuated the inflammatory responses to TNFα compared with the wild-type, whereas these mutations did not alter the responsiveness of these cells to LPS. The T90I mutant macrophages behaved similarly to wild type in response to LPS and TNFα. The TNFR1 levels were increased in whole-cell lysates of TRAPS mutant macrophages, whereas the cell surface expression of TNFR1 was significantly decreased in TRAPS mutant macrophages. Taken together, TRAPS mutations did not augment the inflammatory responses to TNFα and LPS; instead, they suppressed the response to TNFα via decreased cell surface expression of TNFR1. The stimulation of lymphotoxin-α, adenosine triphosphate, and norepinephrine in primary macrophages or various stimuli in murine splenocytes did not induce detectable inflammatory responses. In conclusion, TRAPS mutations suppressed responsiveness to TNFα, and TRAPS-associated inflammation is likely induced by unconfirmed disease-specific proinflammatory factors.

Obesity and Dyslipidemia Synergistically Exacerbate Psoriatic Skin Inflammation.

Patients with psoriasis are frequently complicated with metabolic syndrome; however, it is not fully understood how obesity and dyslipidemia contribute to the pathogenesis of psoriasis. To investigate the mechanisms by which obesity and dyslipidemia exacerbate psoriasis using murine models and neonatal human epidermal keratinocytes (NHEKs), we used wild-type and Apoe-deficient dyslipidemic mice, and administered a high-fat diet for 10 weeks to induce obesity. Imiquimod was applied to the ear for 5 days to induce psoriatic dermatitis. To examine the innate immune responses of NHEKs, we cultured and stimulated NHEKs using IL-17A, TNF-α, palmitic acid, and leptin. We found that obesity and dyslipidemia synergistically aggravated psoriatic dermatitis associated with increased gene expression of pro-inflammatory cytokines and chemokines. Treatment of NHEKs with palmitic acid and leptin amplified pro-inflammatory responses in combination with TNF-α and IL-17A. Additionally, pretreatment with palmitic acid and leptin enhanced IL-17A-mediated c-Jun N-terminal kinase phosphorylation. These results revealed that obesity and dyslipidemia synergistically exacerbate psoriatic skin inflammation, and that metabolic-disorder-associated inflammatory factors, palmitic acid, and leptin augment the activation of epidermal keratinocytes. Our results emphasize that management of concomitant metabolic disorders is essential for preventing disease exacerbation in patients with psoriasis.

SH3BP2 Deficiency Ameliorates Murine Systemic Lupus Erythematosus.

BACKGROUND: The adaptor protein Src homology 3 domain-binding protein 2 (SH3BP2) is widely expressed in immune cells. It controls intracellular signaling pathways. The present study was undertaken to investigate the role of SH3BP2 in a murine systemic lupus erythematosus model. METHODS: For the lupus model, we used Faslpr/lpr mice. Clinical and immunological phenotypes were compared between Faslpr/lpr and SH3BP2-deficient Faslpr/lpr mice. Splenomegaly and renal involvement were assessed. Lymphocyte subsets in the spleen were analyzed by flow cytometry. To examine the role of SH3BP2 in specific cells, B cell-specific SH3BP2-deficient lupus mice were analyzed; T cells and bone marrow-derived dendritic cells and macrophages were analyzed in vitro. RESULTS: SH3BP2 deficiency significantly reduced lupus-like phenotypes, presented as splenomegaly, renal involvement, elevated serum anti-dsDNA antibody, and increased splenic B220+CD4-CD8- T cells. Notably, SH3BP2 deficiency in B cells did not rescue the lupus-like phenotypes. Furthermore, SH3BP2 deficiency did not substantially affect the characteristics of T cells and macrophages in vitro. Interestingly, SH3BP2 deficiency suppressed the differentiation of dendritic cells in vitro and reduced the number of dendritic cells in the spleen of the lupus-prone mice. CONCLUSIONS: SH3BP2 deficiency ameliorated lupus-like manifestations. Modulating SH3BP2 expression could thus provide a novel therapeutic approach to autoimmune diseases.

IL-6-PAD4 axis in the earliest phase of arthritis in knock-in gp130F759 mice, a model for rheumatoid arthritis.

Objective: Animal models for human diseases are especially valuable for clarifying molecular mechanisms before or around the onset. As a model for rheumatoid arthritis (RA), we utilise knock-in mice gp130F759. They have a Y759F mutation in gp130, a common receptor subunit for interleukin 6 (IL-6) family cytokines. Definitive arthritis develops around 8 months old and the incidence reaches 100% around 1 year old. Careful examination in the clinical course revealed very subtle resistance in flexibility of joints at 5 months old. Therefore, pathophysiological changes in gp130F759 were examined to dissect molecular mechanisms for preclinical phase of RA. Methods: Severity of arthritis in gp130F759 was evaluated with a clinical score system and histological quantification. Serum cytokines, autoantibodies and C reactive protein (CRP) were measured. Changes in the synovium were analysed by real-time PCR, flow cytometry and immunohistochemistry. Results: Around 5 months old, various types of cytokines, rheumatoid factor (RF), anti-circular citrullinated peptide IgM and CRP increased in the sera of gp130F759. Enhancement of neovascularisation, synovial hyperplasia and fibrosis was observed. Also, increases in haematopoietic cells dominated by innate immune cells and gene expression of Il6 and Padi4 were detected in the joints. Il6 was expressed by non-haematopoietic synovial cells, whereas PAD4 protein was detected in the synovial neutrophils. Padi4 is induced in neutrophils in vitro by IL-6. Increases of phospho-STAT3 and PAD4 protein were detected in the synovium. Deletion of IL-6 in gp130F759 normalised the amount of PAD4 protein in the joints. Conclusion: The IL-6-PAD4 axis operates in the earliest phase of arthritis in gp130F759, implicating it in early RA.

Sh3bp2 Gain-Of-Function Mutation Ameliorates Lupus Phenotypes in B6.MRL-Faslpr Mice.

SH3 domain-binding protein 2 (SH3BP2) is an adaptor protein that is predominantly expressed in immune cells, and it regulates intracellular signaling. We had previously reported that a gain-of-function mutation in SH3BP2 exacerbates inflammation and bone loss in murine arthritis models. Here, we explored the involvement of SH3BP2 in a lupus model. Sh3bp2 gain-of-function (P416R knock-in; Sh3bp2KI/+) mice and lupus-prone B6.MRL-Faslpr mice were crossed to yield double-mutant (Sh3bp2KI/+Faslpr/lpr) mice. We monitored survival rates and proteinuria up to 48 weeks of age and assessed renal damage and serum anti-double-stranded DNA antibody levels. Additionally, we analyzed B and T cell subsets in lymphoid tissues by flow cytometry and determined the expression of apoptosis-related molecules in lymph nodes. Sh3bp2 gain-of-function mutation alleviated the poor survival rate, proteinuria, and glomerulosclerosis and significantly reduced serum anti-dsDNA antibody levels in Sh3bp2KI/+Faslpr/lpr mice. Additionally, B220+CD4-CD8- T cell population in lymph nodes was decreased in Sh3bp2KI/+Faslpr/lpr mice, which is possibly associated with the observed increase in cleaved caspase-3 and tumor necrosis factor levels. Sh3bp2 gain-of-function mutation ameliorated clinical and immunological phenotypes in lupus-prone mice. Our findings offer better insight into the unique immunopathological roles of SH3BP2 in autoimmune diseases.

Cyclosporin A indirectly attenuates activation of group 2 innate lymphoid cells in papain-induced lung inflammation.

Cyclosporin A (CsA) is a well-known immunosuppressant that is used against steroid-resistant asthma. Group 2 innate lymphoid cells (ILC2s) and type 2 helper T (Th2) cells produce Th2 cytokines including IL-5 and play important roles in asthma pathogenesis. Here, we studied the effects of CsA in allergen-induced lung inflammation in mice and found that CsA decreased the number of lung ILC2s and attenuated papain-induced activation of ILC2s accompanied with IL-5 expression. The ILC2 suppression mediated by CsA was not observed in culture or in lymphocyte-deficient Rag2-/- mice. Thus, we propose a new suppressive effect of CsA, i.e., administration of CsA indirectly suppresses maintenance and activation of lung ILC2s in addition to direct suppression of T-cell activation and cytokine production.

Lnk/Sh2b3 controls the production and function of dendritic cells and regulates the induction of IFN-γ-producing T cells.

Dendritic cells (DCs) are proficient APCs that play crucial roles in the immune responses to various Ags and pathogens and polarize Th cell immune responses. Lnk/SH2B adaptor protein 3 (Sh2b3) is an intracellular adaptor protein that regulates B lymphopoiesis, megakaryopoiesis, and expansion of hematopoietic stem cells by constraining cytokine signals. Recent genome-wide association studies have revealed a link between polymorphism in this adaptor protein and autoimmune diseases, including type 1 diabetes and celiac disease. We found that Lnk/Sh2b3 was also expressed in DCs and investigated its role in the production and function of DC lineage cells. In Lnk(-/-) mice, DC numbers were increased in the spleen and lymph nodes, and growth responses of bone marrow-derived DCs to GM-CSF were augmented. Mature DCs from Lnk(-/-) mice were hypersensitive and showed enhanced responses to IL-15 and GM-CSF. Compared to normal DCs, Lnk(-/-) DCs had enhanced abilities to support the differentiation of IFN-γ-producing Th1 cells from naive CD4(+) T cells. This was due to their elevated expression of IL-12Rß1 and increased production of IFN-γ. Lnk(-/-) DCs supported the appearance of IFN-γ-producing T cells even under conditions in which normal DCs supported induction of regulatory T cells. These results indicated that Lnk/Sh2b3 plays a regulatory role in the expansion of DCs and might influence inflammatory immune responses in peripheral lymphoid tissues.

Lnk prevents inflammatory CD8⁺ T-cell proliferation and contributes to intestinal homeostasis.

The intracellular adaptor Lnk (also known as SH2B3) regulates cytokine signals that control lymphohematopoiesis, and Lnk(-/-) mice have expanded B-cell, megakaryocyte, and hematopoietic stem-cell populations. Moreover, mutations in the LNK gene are found in patients with myeloproliferative disease, whereas LNK polymorphisms have recently been associated with inflammatory and autoimmune diseases, including celiac disease. Here, we describe a previously unrecognized function of Lnk in the control of inflammatory CD8(+) T-cell proliferation and in intestinal homeostasis. Mature T cells from newly generated Lnk-Venus reporter mice had low but substantial expression of Lnk, whereas Lnk expression was downregulated during homeostatic T-cell proliferation under lymphopenic conditions. The numbers of CD44(hi) IFN-γ(+) CD8(+) effector or memory T cells were found to be increased in Lnk(-/-) mice, which also exhibited shortening of villi in the small intestine. Lnk(-/-) CD8(+) T cells survived longer in response to stimulation with IL-15 and proliferated even in nonlymphopenic hosts. Transfer of Lnk(-/-) CD8(+) T cells together with WT CD4(+) T cells into Rag2-deficient mice recapitulated a sign of villous abnormality. Our results reveal a link between Lnk and immune cell-mediated intestinal tissue destruction.

Thymic stromal lymphopoietin (TSLP)-induced polyclonal B-cell activation and autoimmunity are mediated by CD4+ T cells and IL-4.

The cytokine thymic stromal lymphopoietin (TSLP) functions as a regulator of bone marrow B-cell development and a key initiator of allergic inflammation. In the current study, we show that mature B cells, derived from transgenic mice with systemically elevated levels of TSLP (K5-TSLP mice), exhibit markedly enhanced mitogenic responses in vitro and that this enhanced responsiveness leads to polyclonal B-cell activation and development of autoimmune hemolytic anemia in vivo. In contrast, B cells derived from K5-TSLP mice lacking CD4(+) T cells failed to show polyclonal activation. Furthermore, neither mature B-cell activation nor hemolytic anemia occurred in IL-4-deficient K5-TSLP mice. Consistent with these findings, activation of mature B cells occurred independently of B-cell intrinsic TSLP signals. Taken together, our results demonstrate that systemic alterations in TSLP, through induction of IL-4 from CD4(+) T cells and other cell types, functions as an important factor in peripheral B-cell homeostasis and promotion of humoral autoimmunity.

Dibutyl phthalate-induced thymic stromal lymphopoietin is required for Th2 contact hypersensitivity responses.

Thymic stromal lymphopoietin (TSLP) is an IL-7-related cytokine, produced by epithelial cells, that has been linked to atopic dermatitis and asthma; however, it remains unclear how TSLP shapes the adaptive immune response that causes these allergic disorders. In this study, we demonstrate a role for TSLP in a Th2 model of contact hypersensitivity in mice. TSLP is required for the development of Th2-type contact hypersensitivity induced by the hapten FITC in combination with the sensitizing agent dibutyl phthalate. TSLPR-deficient mice exhibited a dramatically reduced response, including markedly reduced local infiltration by eosinophils, Th2 cytokine production, and serum IgE levels, following FITC sensitization and challenge. The reduced response by TSLPR-deficient mice is likely due to decreased frequency and reduced T cell stimulatory function of skin-derived Ag-bearing FITC(+)CD11c(+) dendritic cells in draining lymph nodes following FITC sensitization. These data suggest that skin-derived dendritic cells are direct or indirect targets of TSLP in the development of type 2 immune responses in the skin, where TSLP drives their maturation, accumulation in skin draining lymph nodes, and ability to induce proliferation of naive allergen-specific T cells.

Cutting edge: Inhibition of NF-kappaB-mediated TSLP expression by retinoid X receptor.

The epithelial-derived cytokine thymic stromal lymphopoietin (TSLP) has important roles in the initiation of allergic airway inflammation and the activation of dendritic cells. We have shown that the human TSLP gene is regulated in a NF-kappaB-dependent manner; however the factors that negatively regulate TSLP expression are not known. In this study we demonstrate that 9-cis-retinoic acid (9-cis-RA) is a negative regulator of TSLP expression in airway epithelial cells. This inhibition is manifested as a block in the IL-1beta-mediated recruitment of NF-kappaB to the human TSLP promoter. 9-cis-RA-mediated inhibition is not restricted to TSLP gene expression but rather reflects a general inhibition of NF-kappaB activation, as other NF-kappaB-regulated-genes were also inhibited in a similar manner by 9-cis-RA treatment. Taken as a whole, these data demonstrate that inhibition of IL-1beta-dependent genes by active retinoid X receptors involves antagonism of NF-kappaB signaling.

Local increase in thymic stromal lymphopoietin induces systemic alterations in B cell development.

The cytokine thymic stromal lymphopoietin (TSLP) drives immature B cell development in vitro and may regulate T helper type 2 responses. Here we analyzed the involvement of TSLP in B cell development in vivo with a doxycycline-inducible, keratin 5-driven transgene encoding TSLP (K5-TSLP). K5-TSLP-transgenic mice given doxycycline showed an influx of immature B cells into the periphery, with population expansion of follicular mature B cells, near-complete loss of marginal zone and marginal zone precursor B cells, and 'preferential' population expansion of peritoneal B-1b B cells. These changes promoted cryoglobulin production and immune complex-mediated renal disease. Identical events occurred in mice without T cells, in alternative TSLP-transgenic models and in K5-TSLP-transgenic mice with undetectable systemic TSLP. These observations suggest that signals mediating localized TSLP expression may modulate systemic B cell development and promote humoral autoimmunity.

Differential role of SH2-B and APS in regulating energy and glucose homeostasis.

SH2-B and APS, two members of a pleckstrin homology and SH2 domain-containing adaptor family, promote both insulin and leptin signaling in a similar fashion in cultured cells. In addition, APS mediates insulin-stimulated activation of the c-Cbl/CAP/TC10 pathway in cultured adipocytes. Here we characterized genetically modified mice lacking SH2-B, APS, or both to determine the physiological roles of these two proteins in animals. Disruption of the SH2-B gene resulted in obesity, hyperglycemia, hyperinsulinemia, and glucose intolerance. Conversely, deletion of the APS gene did not alter adiposity, energy balance, and glucose metabolism. Energy intake, energy expenditure, fat content, body weight, and plasma insulin, leptin, glucose, and lipid levels were similar between APS(-/-) and WT littermates fed either normal chow or a high-fat diet. Moreover, deletion of APS failed to alter insulin and glucose tolerance. APS(-/-)/SH2-B(-/-) double knockout mice also developed energy imbalance, obesity, hyperleptinemia, hyperinsulinemia, hyperglycemia, and glucose intolerance; however, plasma leptin and insulin levels were significantly lower in APS(-/-)/SH2-B(-/-) than in SH2-B(-/-) mice. These results suggest that SH2-B, but not APS, is a key positive regulator of energy and glucose metabolism in mice.

Enhanced engraftment of hematopoietic stem/progenitor cells by the transient inhibition of an adaptor protein, Lnk.

Hematopoietic stem cells (HSCs) are the key elements responsible for maintaining blood-cell production throughout life and for lymphohematopoietic reconstitution following bone marrow (BM) transplantation. Enhancement of the engrafting potential and expansion capabilities of HSCs as well as hematopoietic progenitor cells (HPCs) has been a long-time desire as a means of reducing the risks and difficulties that accompany BM transplantation. The ability of HSCs/HPCs to reconstitute the hematopoietic system of irradiated hosts is negatively regulated by an intracellular adaptor protein, Lnk. Here we have identified the functional domains of Lnk and developed a dominant-negative (DN) Lnk mutant that inhibits the functions of Lnk endogenously expressed in the HSCs/HPCs and thereby potentiates the HSCs/HPCs for engraftment. Importantly, even transient expression of DN-Lnk in HSCs/HPCs facilitated their engraftment under nonmyeloablative conditions and fully reconstituted the lymphoid compartments of immunodeficient host animals. HPCs expressing DN-Lnk were efficiently trapped by immobilized vascular cell adhesion molecule-1 (VCAM-1) in a transwell migration assay, suggesting involvement of Lnk in the regulation of cell mobility or cellular interaction in microenvironments. Transient inhibition of Lnk or Lnk-mediated pathways could be a potent approach to augment engraftment of HSCs/HPCs without obvious side effects.

APS, an adaptor molecule containing PH and SH2 domains, has a negative regulatory role in B cell proliferation.

Adaptor molecule containing PH and SH2 domains (APS) is an intracellular adaptor protein that forms part of an adaptor family along with Lnk and SH2-B. APS transcripts are expressed in various tissues including brain, kidney, and muscle, as well as in splenic B cells but not in T cells. We investigated the functions of APS in B cell development and activation by generating APS-transgenic (APS-Tg) mice that overexpressed APS in lymphocytes. The number of B-1 cells in the peritoneal cavity was reduced in APS-Tg mice, as were B-2 cells in the spleen. B cell development in the bone marrow was partially impaired at the transition stage from proliferating large pre-B to small pre-B cells. B cell proliferation induced by B cell receptor (BCR) crosslinking but not by other B cell mitogens was also impaired in APS-Tg mice. APS co-localized with BCR complexes and filamentous actin in activated APS-Tg B cells. Thus, APS appears to play novel negative regulatory roles in BCR signaling, actin reorganization pathways, and control of compartment sizes of B-lineage cells.

Increased numbers of B-1 cells and enhanced responses against TI-2 antigen in mice lacking APS, an adaptor molecule containing PH and SH2 domains.

APS (adaptor molecule containing PH and SH2 domains) is an intracellular adaptor protein that forms an adaptor family along with Lnk and SH2-B. While experiments using cultured cell lines have demonstrated that APS is phosphorylated in response to various stimuli, its in vivo functions remain unclear. We attempted to determine the physiological roles of APS by generating APS-deficient (APS(-/-)) mice. APS(-/-) mice were viable and fertile and showed no abnormalities or growth retardation. Immunologically, APS(-/-) mice showed normal development and distribution of lymphocytes and myeloid cells, except for increased numbers of B-1 cells in the peritoneal cavity. APS(-/-) mice exhibited an enhanced humoral immune response against trinitrophenol-Ficoll, a thymus-independent type 2 antigen, while APS(-/-) B-2 cells exhibited normal proliferative responses and tyrosine phosphorylation of intracellular proteins upon B-cell receptor (BCR) cross-linking. APS colocalized with filamentous actin (F-actin) accumulated during the capping of BCRs in APS-transgenic B cells. After BCR stimulation, F-actin contents were lower in APS(-/-) B-1 cells than in wild-type B-1 cells. Our results indicate that APS might have a novel regulatory role in actin reorganization and control of B-1 cell compartment size.

Roles of a conserved family of adaptor proteins, Lnk, SH2-B, and APS, for mast cell development, growth, and functions: APS-deficiency causes augmented degranulation and reduced actin assembly.

Lnk, SH2-B, and APS form a conserved adaptor protein family. All of those proteins are expressed in mast cells and their possible functions in signaling through c-Kit or FcRI have been speculated. To investigate roles of Lnk, SH2-B or APS in mast cells, we established IL-3-dependent mast cells from Ink-/-, SH2-B-/-, and APS -/- mice. IL-3-dependent growth of those cells was comparable. Proliferation or adhesion mediated by c-Kit as well as degranulation induced by cross-linking FcRI were normal in the absence of Lnk or SH2-B. In contrast, APS-deficient mast cells showed augmented degranulation after cross-linking FcRI compared to wild-type cells, while c-Kit-mediated proliferation and adhesion were kept unaffected. APS-deficient mast cells showed reduced actin assembly at steady state, although their various intracellular responses induced by cross-linking FcRI were indistinguishable compared to wild-type cells. Our results suggest potential roles of APS in controlling actin cytoskeleton and magnitude of degranulation in mast cells.