Toll-like receptors (TLRs) play a critical role in stimulating innate immunity by recognizing pathogen-associated molecular patterns (PAMPs) on invading microorganisms. Platelets also play a role in innate immunity, and we studied whether they express TLR. Results show that human and murine platelets variably expressed TLR2, TLR4, and TLR9 by flow cytometry and Western blotting.

SummaryBeginning at the time of insulitis (7 wk of age), CD4 + and CD8 + mature thymocytes from nonobese diabetic (NOD) mice exhibit a proliferative unresponsiveness in vitro after T cell receptor (TCR) crosslinking. This unresponsiveness does not result from either insulitis or thymic involution and is long lasting, i.e., persists until diabetes onset (24 wk of age). We previously proposed that it represents a form of thymic T cell anergy that predisposes to diabetes onset. This hypothesis was tested in the present study by further investigating the mechanism responsible for NOD thymic T cell proliferative unresponsiveness and determining whether reversal of this unresponsiveness protects NOD mice from diabetes. Interleukin 4 (IL-4) secretion by thymocytes from >7-wk-old NOD mice was virtually undetectable after treatment with either anti-TCR c~/B, anti-CD3, or Concanavalin A (Con A) compared with those by thymocytes from age-and sexmatched control BALB/c mice stimulated under identical conditions. NOD thymocytes stimulated by anti-TCR o~/B or anti-CD3 secreted less IL-2 than did similarly activated BALB/c thymocytes. However, since equivalent levels of IL-2 were secreted by Con A-activated NOD and BALB/c thymocytes, the unresponsiveness of NOD thymic T cells does not appear to be dependent on reduced IL-2 secretion. The surface density and dissociation constant of the high affinity IL-2 receptor of Con A-activated thymocytes from both strains are also similar. The patterns of unresponsiveness and lymphokine secretion seen in anti-TCR/CD3-activated NOD thymic T cells were also observed in activated NOD peripheral spleen T cells. Exogenous recombinant (r)IL-2 only partially reverses NOD thymocyte proliferative unresponsiveness to anti-CD3, and this is mediated by the inability of IL-2 to stimulate a complete IL-4 secretion response. In contrast, exogenous rlL-4 reverses the unresponsiveness of both NOD thymic and peripheral T cells completely, and this is associated with the complete restoration of an IL-2 secretion response. Furthermore, the in vivo administration of rlL-4 to prediabetic NOD mice protects them from diabetes. Thus, the ability of rlL-4 to reverse completely the NOD thymic and peripheral T cell proliferative defect in vitro and protect against diabetes in vivo provides further support for a causal relationship between this T cell proliferative unresponsiveness and susceptibility to diabetes in NOD mice.

To determine whether inhibition of Syk would be useful in Fc␥R-dependent cytopenias such as immune thrombocytopenic purpura (ITP) or autoimmune hemolytic anemia, mouse models were used to evaluate efficacy of R406, an inhibitor of Syk function, in treating cytopenia. Both disease models responded favorably to treatment, with amelioration of ITP being more dramatic. Thus, phase 2 clinical trial was initiated to study the effects of Syk inhibition in humans with ITP. Sixteen adults with chronic ITP were entered into an open-label, single-arm cohort doseescalation trial beginning with 75 mg and escalating as high as 175 mg twice daily. Doses were increased until a persistent response was seen, toxicity occurred, or 175 mg twice daily was reached. Eight patients achieved persistent responses with platelet counts greater than 50 ؋ 10 9 /L (50 000 mm 3 ) on more than 67% (actually 95%) of their study visits, including 3 who had not persistently responded to thrombopoietic agents. Four others had nonsustained responses. Mean peak platelet count exceeded 100 ؋ 10 9 /L (100 000 mm 3 ) in these 12 patients. Toxicity was primarily GI-related with diarrhea (urgency) and vomiting; 2 patients had transaminitis.

Despite a more than 20-year experience of therapeutic benefit, the relevant molecular and cellular targets of intravenous immunoglobulin (IVIg) in autoimmune disease remain unclear. Contrary to the prevailing theories of IVIg action in autoimmunity, we show that IVIg drives signaling through activating Fc gamma receptors (Fc gammaR) in the amelioration of mouse immune thrombocytopenic purpura (ITP). The actual administration of IVIg was unnecessary because as few as 10(5) IVIg-treated cells could, upon adoptive transfer, ameliorate ITP. IVIg did not interact with the inhibitory Fc gammaRIIB on the initiator cell, although Fc gammaRIIB does have a role in the late phase of IVIg action. Notably, only IVIg-treated CD11c+ dendritic cells could mediate these effects. We hypothesize that IVIg forms soluble immune complexes in vivo that prime dendritic-cell regulatory activity. In conclusion, the clinical effects of IVIg in ameliorating ITP seem to involve the acute interaction of IVIg with activating Fc gammaR on dendritic cells.

Immune thrombocytopenia (ITP) is a bleeding disorder characterized by antibodyopsonized platelets being prematurely destroyed in the spleen, although some patients with ITP may have a cell-mediated form of thrombocytopenia. Although several animal models of ITP have been developed, few mimic primary chronic ITP nor have any shown cell-mediated platelet destruction. To create this type of model, splenocytes from CD61 knockout mice immunized against CD61 ؉ platelets were transferred into severe combined immunodeficient (SCID) (CD61 ؉ ) mouse recipients, and their platelet counts and phenotypes were observed. As few as 5 ؋ 10 4 splenocytes induced a significant thrombocytopenia and bleeding mortality (80%) in recipients within 3 weeks after transfer. Depletion of lymphocyte subsets before transfer showed that the splenocyte's ability to induce thrombocytopenia and bleeding completely depended on CD4 ؉ T helper cells and that both CD19 ؉ B cell (antibody)-and CD8 ؉ T cell (cell)-mediated effector mechanisms were responsible. Treatment of the SCID mouse recipients with intravenous ␥-globulins raised platelet counts and completely prevented bleeding mortality induced by antibodymediated effector mechanisms but did not affect cell-mediated disease. This novel model not only shows both antibody-and cell-mediated ITP and bleeding but also suggests that these 2 effector mechanisms have a differential response to therapy. (Blood. 2010;115:1247-1253) IntroductionImmune thrombocytopenia (ITP), one of the most common hematologic autoimmune bleeding disorders, is characterized by premature platelet clearance by Fc␥ receptor (Fc␥R)-mediated phagocytosis in the reticuloendothelial system. 1-8 ITP was distinguished by 2 forms, termed acute and chronic, but an international group of recognized experts has significantly revised the definitions of and recommendations for the clinical diagnosis of ITP. 8 Primary ITP is now defined as an autoimmune disorder characterized by isolated thrombocytopenia (peripheral blood platelet count Ͻ 100 ϫ 10 9 /L) in the absence of other causes or disorders that may be associated with thrombocytopenia. 8 The various phases of ITP are defined by the time since diagnosis; newly diagnosed ITP occurs within 3 months from diagnosis, whereas persistent ITP is between 3 and 12 months from diagnosis, and chronic ITP is now defined as thrombocytopenia lasting for more than 12 months. 8 The classification of severe ITP is now reserved for those patients in whom there is the presence of bleeding symptoms at presentation or the occurrence of new bleeding symptoms requiring therapeutic intervention. 8 First-line treatments for patients with chronic ITP include steroids and intravenous ␥-globulins (IVIgs), and previous studies have shown that IVIg can also protect against passive ITP in mice. [9][10][11][12] At least 70% of patients with chronic ITP have identifiable serum autoantibodies that are composed primarily of IgG1 and IgG3 isotypes and generally target platelet glycoproteins (GPs) IIb/IIIa and/or GPIbIX. ...

High-dose i.v. Ig (IVIG) is used to treat various autoimmune and inflammatory diseases; however, the mechanism of action remains unclear. Based on the K/BxN serum transfer arthritis model in mice, IVIG suppression of inflammation has been attributed to a mechanism involving basophils and the binding of highly sialylated IgG Fc to DC-SIGN–expressing myeloid cells. The requirement for sialylation was examined in the collagen Ab-induced arthritis (CAbIA) and K/BxN serum transfer arthritis models in mice. High-dose IVIG (1–2 g/kg body weight) suppressed inflammatory arthritis when given prophylactically. The same doses were also effective in the CAbIA model when given subsequent to disease induction. In this therapeutic CAbIA model, the anti-inflammatory effect of IVIG was dependent on IgG Fc but not F(ab′)2 fragments. Removal of sialic acid residues by neuraminidase had no impact on the anti-inflammatory activity of IVIG or Fc fragments. Treatment of mice with basophil-depleting mAbs did not abrogate the suppression of either CAbIA or K/BxN arthritis by IVIG. Our data confirm the therapeutic benefit of IVIG and IgG Fc in Ab-induced arthritis but fail to support the significance of sialylation and basophil involvement in the mechanism of action of IVIG therapy.

Summary. Although the mechanism of action of intravenous immunoglobulin (IVIg) in treating antibody-dependent thrombocytopenia remains unclear, most studies have suggested that IVIg blocks the function of Fc receptors in the reticuloendothelial system (RES) and/or the protective effect may be due to the presence of variable region-reactive (anti-idiotype) antibodies within IVIg. We evaluated the effect of IVIg on platelet counts in a murine model of passively induced immune thrombocytopenia (PIT). Although IVIg was unable to neutralize the binding of two platelet-specific monoclonal antibodies to their target antigens either in vivo or in vitro, it was able to prevent PIT as well as ameliorate pre-established PIT mediated by these antibodies. IVIg adsorbed against the antibody used to induce thrombocytopenia or endogenous murine immunoglobulin also protected against PIT, indicating that antibodies with anti-idiotype activity present in IVIg are not necessary for its effective treatment of PIT. IVIg significantly blocked the ability of the RES to clear antibody-sensitized red blood cells. F(ab H ) 2 fragments of IVIg, which are unable to block the RES but retain the idiotypic regions, were ineffective at protecting mice from PIT. Our data suggest that IVIg exerts its rapid effect by inhibiting RES function and that anti±idiotype interactions are not required.

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