Seng Song

INTRAVENOUS IMMUNOGLOBULIN AMELIORATES ITP VIA ACTIVATING FC GAMMA RECEPTORS ON DENDRITIC CELLS

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 Fcγ receptors (FcγR) in the amelioration of mouse immune thrombocytopenic purpura (ITP). The actual administration of IVIg was unnecessary because as few as 105 IVIg-treated cells could, upon adoptive transfer, ameliorate ITP. IVIg did not interact with the inhibitory FcγRIIB on the initiator cell, although FcγRIIB 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 FcγR on dendritic cells.

IVIg inhibits reticuloendothelial system function and ameliorates murine passive-immune thrombocytopenia independent of anti-idiotype reactivity.

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′)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.

Can antibodies with specificity for soluble antigens mimic the therapeutic effects of intravenous IgG in the treatment of autoimmune disease

Intravenous Ig (IVIg) mediates protection from the effects of immune thrombocytopenic purpura (ITP) as well as numerous other autoimmune states; however, the active antibodies within IVIg are unknown. There is some evidence that antibodies specific for a cell-associated antigen on erythrocytes are responsible, at least in part, for the therapeutic effect of IVIg in ITP. Yet whether an IVIg directed to a soluble antigen can likewise be beneficial in ITP or other autoimmune diseases is also unknown. A murine model of ITP was used to determine the effectiveness of IgG specific to soluble antigens in treating immune thrombocytopenic purpura. Mice experimentally treated with soluble OVA + anti-OVA versus mice treated with OVA conjugated to rbcs (OVA-rbcs) + anti-OVA were compared. In both situations, mice were protected from ITP. Both these experimental therapeutic regimes acted in a complement-independent fashion and both also blocked reticuloendothelial function. In contrast to OVA-rbcs + anti-OVA, soluble OVA + anti-OVA (as well as IVIg) did not have any effect on thrombocytopenia in mice lacking the inhibitory receptor FcgammaRIIB (FcgammaRIIB(-/-) mice). Similarly, antibodies reactive with the endogenous soluble antigens albumin and transferrin also ameliorated ITP in an FcgammaRIIB-dependent manner. Finally, broadening the significance of these experiments was the finding that anti-albumin was protective in a K/BxN serum-induced arthritis model. We conclude that IgG antibodies directed to soluble antigens ameliorated 2 disparate IVIg-treatable autoimmune diseases.

Mechanisms of action of intravenous immunoglobulin in the treatment of immune thrombocytopenia

Intravenous immunoglobulin (IVIG) is currently used to treat a multitude of autoimmune disorders including immune thrombocytopenic purpura (ITP), yet the mechanism of action of IVIG remains unresolved. Using a murine model of ITP in which IVIG functions therapeutically, our laboratory has addressed such theories as blockade/inhibition of the mononuclear phagocytic system, cytokine regulation, and neutralization of pathogenic autoantibodies mediated by anti‐idiotypic antibodies, and these findings will be discussed herein. We have also demonstrated that soluble immune complexes can completely recapitulate the therapeutic effects of IVIG in ITP, and recent work from us has identified activating Fcγ receptors on CD11c+ dendritic cells as the relevant molecular target of IVIG in the acute resolution of murine immune thrombocytopenia. This and other work to devise antibody‐based IVIG alternative therapies will also be addressed. Pediatr Blood Cancer 2006;47:710–713.

Porcine von Willebrand factor and thrombin induce the activation of c-Jun amino-terminal kinase (JNK/SAPK) whereas only thrombin induces activation of extracellular signal-related kinase 2 (ERK2) in human platelets

The interaction of platelets with subendothelial von Willebrand factor (VWF), especially under high shear stress, is considered to be the first activation step which primes platelets for subsequent haemostatic events. The signalling cascade which results from the interaction of VWF and its receptor GPIbIX has only been partially defined. Mitogen‐activated protein kinases (MAPKs) are a family of downstream transmembrane signalling serine–threonine kinases and have been demonstrated to be present and functional in platelets; these include the extracellular signal‐related kinases (ERKs), c‐Jun amino‐terminal kinases (JNKs) and p38 MAPK. Previously, we showed that p38 MAPK was not required in VWF‐induced human platelet activation. It is not known whether VWF‐dependent platelet activation involves the activation of the JNK and ERK family of signalling molecules. This report demonstrates that porcine von Willebrand factor (pVWF) induced a sustained and stable JNK activation measurable by 1 min after activation. Thrombin also induced JNK activation assessed at 1 min after activation. In contrast to thrombin, pVWF did not induce ERK2 activation at any time point tested. To ensure that ERK activation was unnecessary for pVWF‐dependent platelet activation, we functionally inhibited ERK‐dependent signalling with PD98059, a potent and selective inhibitor of the MAP kinase kinase (MEK‐1), which is the upstream kinase of ERK1 and ERK2. Although PD98059 inhibited ERK2 activation in platelets, it had no effect on pVWF‐ or thrombin‐induced platelet alpha or lysozomal granule release, modulation of membrane glycoprotein CD41, microparticle formation, platelet shape change or platelet agglutination. It is concluded that pVWF and thrombin induced JNK activation, but whereas thrombin induced ERK2 activation VWF did not; functional ERK2 activity was also not required for pVWF‐ or thrombin‐dependent platelet activation.

p38 MAPK is activated but not necessary in porcine von Willebrand factor-dependent platelet activation

We have investigated the role of p38 mitogen‐activated protein kinase (MAPK) in von Willebrand factor (VWF)‐dependent platelet activation. The interaction of platelets with subendothelial VWF, especially under high shear stress, is considered to be the first activation step which primes platelets for subsequent haemostatic events. As a model of VWF‐dependent platelet activation, porcine VWF was employed. Porcine VWF induced p38 MAPK activation by 1 min post‐addition; assessed by phosphorylation of a recombinant p38 MAPK fusion protein substrate termed glutathione S‐transferase‐MAPK activated protein kinase‐2. To determine if p38 MAPK was necessary for porcine VWF‐induced platelet activation, we functionally inhibited p38 MAPK activity with SB203580 before exposure of the platelets to porcine VWF. Inhibition of p38 MAPK had no effect on VWF‐induced platelet alpha or lysozomal granule release, expression of activated GPIIb IIIa, modulation of membrane glycoprotein CD41, expression of phosphatidylserine as assessed by annexin V binding, microparticle formation, or platelet agglutination. It was concluded that SB203580‐inhibitable p38 MAPK activity induced by porcine VWF is not necessary for platelet activation.

von Willebrand factor (VWF)-dependent human platelet activation: porcine VWF utilizes different transmembrane signaling pathways than does thrombin to activate platelets, but both require protein phosphatase function

Summary.  The interaction between von Willebrand factor (VWF) and glycoprotein (GP) Ib results in platelet agglutination and activation of many signaling intermediates. To determine if VWF‐dependent platelet activation requires the participation of pivotal transmembrane signaling pathways, we analyzed VWF‐dependent platelet activation profiles following inhibition of several transmembrane signaling intermediates. This was accomplished using porcine VWF, which has been shown to interact with human GPIb independently of shear stress or ristocetin. Platelet alpha (CD62) and lysozomal granule release (CD63), microparticle formation, and platelet agglutination/aggregation were evaluated. The ability of signaling inhibitors to prevent VWF‐dependent platelet activation was compared to their ability to inhibit thrombin‐dependent activation. The results demonstrate that VWF‐dependent platelet activation can occur independently of the activities of protein kinase C (PKC), wortmannin‐sensitive phosphatidylinositide 3‐kinase, and phospholipase C, as well as independently of elevations in the concentration of intracellular calcium. In sharp contrast, these transmembrane signaling intermediates are required for thrombin‐dependent platelet activation. In addition, thrombin‐dependent but not VWF‐dependent platelet activation was associated with elevations in the concentration of intracellular calcium under the conditions used. The family of signaling intermediates which appeared to be pivotal for both thrombin‐ and VWF‐dependent platelet activation were the protein tyrosine phosphatases and the serine/threonine phosphatases. It is concluded that thrombin‐dependent platelet activation relies on the activation of several transmembrane signaling pathways, whereas VWF‐dependent platelet activation is dependent upon the activity of protein phosphatases. Inhibition of these phosphatases in vivo may provide a novel therapeutic approach for treating VWF‐dependent platelet disorders such as thrombotic thrombocytopenic purpura or arterial thrombosis.