Anti-HLA antibodies with complementary and synergistic interaction geometries promote classical complement activation on platelets.

High titers of HLA antibodies are associated with platelet refractoriness, causing poor platelet increments after transfusions in a subset of patients with HLA antibodies. Currently, we do not know the biological mechanisms that explain the variability in clinical responses in HLA alloimmunized patients receiving platelet transfusions. Previously we showed that a subset of anti-HLA IgG-antibodies induces FcγRIIa-dependent platelet activation and enhanced phagocytosis. Here, we investigated whether anti-HLA IgG can induce complement activation on platelets. We found that a subset of anti-HLA IgG induced complement activation via the classical pathway, causing C4b and C3b deposition and formation of the membrane-attack complex. This resulted in permeabilization of platelet membranes and increased calcium influx. Complement activation also caused enhanced α-granule release, as measured by CD62P surface exposure. Blocking studies revealed that platelet activation was caused by FcγRIIa-dependent signaling as well as HLA antibody induced complement activation. Synergistic complement activation employing combinations of monoclonal IgGs suggested that assembly of oligomeric IgG complexes strongly promoted complement activation through binding of IgGs to different antigenic determinants on HLA. In agreement with this, we observed that preventing anti-HLA-IgG hexamer formation using an IgG-Fc:Fc blocking peptide, completely inhibited C3b and C4b deposition. Our results show that HLA antibodies can induce complement activation on platelets including membrane attack complex formation, pore formation and calcium influx. We propose that these events can contribute to fast platelet clearance in vivo in patients refractory to platelet transfusions with HLA alloantibodies, who may benefit from functional-platelet matching and treatment with complement inhibitors.

Inhibition of recall responses through complementary therapies targeting CD8+ T-cell- and alloantibody-dependent allocytotoxicity in sensitized transplant recipients.

Allospecific T memory cell responses in transplant recipients arise from environmental exposure to previous transplantation or cross-reactive heterologous immunity. Unfortunately, these memory responses pose a significant barrier to the survival of transplanted tissue. We have previously reported that concurrent inhibition of CD154 and LFA-1 suppresses primary CD8-dependent rejection responses that are not controlled by conventional immunosuppressive strategies. We hypothesized that CD154- and LFA-1-mediated inhibition, by targeting activation as well as effector functions, may also be efficacious for the control of alloreactive CD8+ T-cell responses in sensitized hosts. We found that treatment with anti-LFA-1 mAb alone enhanced transplant survival and reduced CD8-mediated cytotoxicity in sensitized CD4 KO recipients. However, treatment with anti-CD154 mAb alone did not have an effect. Notably, when both CD4- and CD8-dependent rejection pathways are operative (wild-type sensitized recipients), LFA-1 significantly inhibited CD8-mediated in vivo allocytotoxicity but did not correspond with enhanced hepatocyte survival. We hypothesized that this was due to alloantibody-mediated rejection. When anti-LFA-1 mAb treatment was combined with macrophage depletion, which we have previously reported impairs alloantibody-mediated parenchymal cell damage, in vivo cytotoxic effector function was significantly decreased and was accompanied by significant enhancement of hepatocyte survival in sensitized wild-type recipients. Therefore, LFA-1 is a potent therapeutic target for reduction of CD8-mediated cytotoxicity in sensitized transplant recipients and can be combined with other treatments that target non-CD8-mediated recall alloimmunity.

NO mediates antifibrotic actions of L-arginine supplementation following induction of anti-thy1 glomerulonephritis.

UNLABELLED: NO mediates antifibrotic actions of L-arginine supplementation following induction of anti-thy1 glomerulonephritis. BACKGROUND: L-Arginine plays a complex role in renal matrix expansion, involving endogenous metabolism into nitric oxide (NO), polyamines, L-proline and agmatine. Supplementing dietary L-arginine intake has been shown to limit transforming growth factor (TGF)-beta 1 overproduction and matrix accumulation in rats with induced anti-thy1 glomerulonephritis (GN). The present study tests the hypothesis that this beneficial effect on in vivo TGF-beta overexpression is mediated via the generation of NO. METHODS: One day after induction of anti-thy1 GN, male Wistar rats fed a normal protein diet were assigned to the following groups: (1) normal controls; (2) GN; (3) GN-Arg (plus 500 mg L-arginine/day); (4) GN-Arg-NAME [plus 500 mg L-arginine/day and 75 mg/L of the NO synthase inhibitor nitro-L-arginine-methyl ester (L-NAME) in the drinking water]; and (5) GN-Molsi (10 mg/day of the NO donor molsidomine). In protocol 1, treatment lasted until day 7, and in protocol 2, until day 12 after disease induction, respectively. Analysis included systolic blood pressure, a glomerular histologic matrix score, and the glomerular mRNA and protein expression of the key fibrogen TGF-beta1, the matrix protein fibronectin, and the protease inhibitor plasminogen activator inhibitor type 1 (PAI-1). RESULTS: Blood pressure was normal in untreated anti-thy1 animals and not significantly affected by any of the treatments. Compared to untreated nephritic rats, administration of both L-arginine and molsidomine reduced glomerular TGF-beta 1 overexpression significantly and to a similar degree in both protocols, while the beneficial effect of L-arginine was abolished by concomitant NO synthesis inhibition. Glomerular matrix accumulation, fibronectin and PAI-1 mRNA and protein expression closely followed the expression of TGF-beta 1. CONCLUSION: The present study shows that L-arginine's antifibrotic action in normotensive anti-thy1 GN is mainly mediated by endogenous production of NO. The data suggest that NO limits in vivo TGF-beta overexpression in a pressure-independent manner and that NO donors may be of benefit in the treatment of human fibrotic renal disease.

Functional in vivo characterization of human monoclonal anti-D in NOD-scid mice.

The prophylaxis of the hemolytic disease of the newborn requires significant amounts of plasma-derived polyclonal human anti-D. Because of procurement problems, there is a growing interest in replacing plasma-derived anti-D by in vitro-produced human monoclonal anti-D. Hundreds of monoclonal anti-D have been prepared, but the selection of the most potent for in vivo use is difficult because it cannot be predicted by in vitro characterization. This study evaluated the possibility of using nonobese diabetic/severe combined immunodeficient (NOD-scid) mice for the in vivo evaluation of human monoclonal anti-D. Human red blood cells (RBCs) were found to circulate normally in the blood of NOD-scid mice previously injected with a physiologic amount of human immunoglobulin G (10 mg). The addition of a small amount of anti-D (1 to 5 microg) resulted in the clearance of Rh D(+) RBCs within 4 hours. The comparative testing of 8 monoclonal anti-Ds showed a wide range of potency (15% to 87%) relative to plasma-derived polyclonal anti-D. There was no strong correlation between the in vivo potency index and the immunoglobulin G isotype, affinity, or fine specificity of the antibodies. These results show the usefulness of NOD-scid mice for the initial in vivo screening of human monoclonal anti-D before testing the most active antibodies in clinical trials done in human volunteers.