Florian Länger

Anti-CD40L Immune Complexes Potently Activate Platelets In Vitro and Cause Thrombosis in FCGR2A Transgenic Mice

Anti-CD40L immunotherapy in systemic lupus erythematosus patients was associated with thromboembolism of unknown cause. We previously showed that monoclonal anti-CD40L immune complexes (ICs) activated platelets in vitro via the IgG receptor (FcγRIIa). In this study, we examined the prothrombotic effects of anti-CD40L ICs in vivo. Because mouse platelets lack FcγRIIa, we used FCGR2A transgenic mice. FCGR2A mice were injected i.v. with preformed ICs consisting of either anti-human CD40L mAb (M90) plus human CD40L, or a chimerized anti-mouse CD40L mAb (hMR1) plus mouse CD40L. ICs containing an aglycosylated form of hMR1, which does not bind FcγRIIa, were also injected. M90 IC caused shock and thrombocytopenia in FCGR2A but not in wild-type mice. Animals injected with hMR1 IC also experienced these effects, whereas those injected with aglycosylated-hMR1 IC did not, demonstrating that anti-CD40L IC-induced platelet activation in vivo is FcγRIIa-dependent. Sequential injections of individual IC components caused similar effects, suggesting that ICs were able to assemble in circulation. Analysis of IC-injected mice revealed pulmonary thrombi consisting of platelet aggregates and fibrin. Mice pretreated with a thrombin inhibitor became moderately thrombocytopenic in response to anti-CD40L ICs and had pulmonary platelet-thrombi devoid of fibrin. In conclusion, we have shown for the first time that anti-CD40L IC-induced thrombosis can be replicated in mice transgenic for FcγRIIa. This molecular mechanism may be important for understanding thrombosis associated with CD40L immunotherapy. The FCGR2A mouse model may also be useful for assessing the hemostatic safety of other therapeutic Abs.

Lung-Specific Overexpression of CC Chemokine Ligand (CCL) 2 Enhances the Host Defense to Streptococcus pneumoniae Infection in Mice: Role of the CCL2-CCR2 Axis

Mononuclear phagocytes are critical components of the innate host defense of the lung to inhaled bacterial pathogens. The monocyte chemotactic protein CCL2 plays a pivotal role in inflammatory mononuclear phagocyte recruitment. In this study, we tested the hypothesis that increased CCL2-dependent mononuclear phagocyte recruitment would improve lung innate host defense to infection with Streptococcus pneumoniae. CCL2 transgenic mice that overexpress human CCL2 protein in type II alveolar epithelial cells and secrete it into the alveolar air space showed a similar proinflammatory mediator response and neutrophilic alveolitis to challenge with S. pneumoniae as wild-type mice. However, CCL2 overexpressing mice showed an improved pneumococcal clearance and survival compared with wild-type mice that was associated with substantially increased lung mononuclear phagocyte subset accumulations upon pneumococcal challenge. Surprisingly, CCL2 overexpressing mice developed bronchiolitis obliterans upon pneumococcal challenge. Application of anti-CCR2 Ab MC21 to block the CCL2-CCR2 axis in CCL2 overexpressing mice, though completely abrogating bronchiolitis obliterans, led to progressive pneumococcal pneumonia. Collectively, these findings demonstrate the importance of the CCL2-CCR2 axis in the regulation of both the resolution/repair and remodelling processes after bacterial challenge and suggest that overwhelming innate immune responses may trigger bronchiolitis obliterans formation in bacterial lung infections.

Apoptosis of regulatory T lymphocytes is increased in chronic inflammatory bowel disease and reversed by anti-TNFα treatment

Background and aims Inappropriate immune responses contribute to the continuous stimulation of the intestinal immune system in chronic inflammatory bowel disease (IBD). Among several pathogenic factors, a numerical deficiency of regulatory T (Treg) cells has been suggested to lead to an insufficient compensation of chronically activated T lymphocytes. This study was conducted to investigate whether increased apoptosis contributes to Treg cell deficiency in IBD and whether successful treatment with antitumour necrosis factor α (TNFα) is achieved by reducing of Treg cell apoptosis. Methods Apoptosis of CD4+Foxp3+ Treg cells in tissue sections of patients with active IBD was analysed by immunohistochemistry and TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labelling) staining. Apoptosis of peripheral blood CD4+CD25+Foxp3+ Treg cells was investigated by flow cytometry and annexin-V staining. In addition, caspase activity and apoptosis were measured in sera of patients with IBD treated with anti-TNFα by a luminometric caspase enzyme assay. Results It is demonstrated that patients with active IBD revealed increased apoptosis of local CD4+Foxp3+ Treg cells in the inflamed mucosa compared with non-inflamed control colon tissue. Moreover, in peripheral blood a reduced frequency and increased apoptosis of Treg cells were found and accompanied by elevated caspase activity in the serum. During anti-TNFα treatment, Treg cell apoptosis declined in close correlation with elevated peripheral Treg cell numbers and a decrease of caspase activation and disease activity. Conclusions These data suggest that increased apoptosis of Treg cells plays a potentially important role in the pathogenesis of IBD and can be reversed by anti-TNFα treatment. Measurement of Treg cell apoptosis and serum caspase activity might therefore represent promising tools for monitoring disease activity and treatment response in patients with IBD.

The role of CD40 in CD40L- and antibody-mediated platelet activation

Our initial finding that CD40- and CD40 ligand (CD40L)-deficient mice displayed prolonged tail bleeding and platelet function analyzer (PFA-100) closure times prompted us to further investigate the role of the CD40-CD40L dyad in primary hemostasis and platelet function. Recombinant human soluble CD40L (rhsCD40L), chemical cross-linking of which suggested a trimeric structure of the protein in solution, activated platelets in a CD40-dependent manner as evidenced by increased CD62P expression. CD40 monoclonal antibody (mAb) M3, which completely blocked rhsCD40L-induced platelet activation, also prolonged PFA-100 closure times of normal human blood. In contrast, CD40 mAb G28-5 showed less potential in blocking rhsCD40L-induced CD62P expression and did not affect PFA-100 closure times. However, when added to the platelets after rhsCD40L, G28-5 significantly enhanced the platelet response by causing clustering of, and signaling through, FcgammaRII. Similarly, higher order multimeric immune complexes formed at a 1/3 molar ratio of M90, a CD40L mAb, to rhsCD40L induced strong Fcgamma RII-mediated platelet activation when translocated to the platelet surface in a CD40-dependent manner, including the induction of morphological shape changes, fibrinogen binding, platelet aggregation, dense granule release, microparticle generation and monocyte-platelet-conjugate formation. The results suggest that CD40 may play a role in primary hemostasis and platelet biology by two independent mechanisms: First, by functioning as a primary signaling receptor for CD40L and, second, by serving as a docking molecule for CD40L immune complexes. The latter would also provide a potential mechanistic explanation for the unexpected high incidence of CD40L mAb-associated thrombotic events in recent human and animal studies.

Tissue factor procoagulant activity of plasma microparticles in patients with cancer-associated disseminated intravascular coagulation.

Tissue factor (TF) expressed on sub-cellular membrane vesicles, so-called plasma microparticles (MPs), has recently emerged as a potential key player in intravascular coagulation activation in various disease states. In this report, we demonstrate significantly increased levels of TF-specific procoagulant activity (PCA) of plasma MPs in five patients presenting with overt disseminated intravascular coagulation (DIC) due to an underlying malignancy, including non-small-cell lung cancer (n = 1), melanoma (n = 1), prostate cancer (n = 2), and acute promyelocytic leukemia (n = 1). Clotting experiments on available tumor cell samples suggested that cancer cells were a potential source of circulating TF-positive MPs at least in three of the five patients. Furthermore, follow-up plasma samples from two surviving patients revealed that response of their malignancies to specific anti-cancer therapy was paralleled by resolution of overt DIC and a significant decline in MP-associated TF PCA. Levels of plasma TF antigen, as assessed by an enzyme-linked immunosorbent assay, were also increased at presentation albeit to a lesser extent compared to MP-associated TF PCA, likely due to insufficient solubilization of the phospholipid-incorporated full-length TF molecule by the detergent. In summary, our findings suggest that MP-associated TF PCA may play an important pathogenic role in the evolution of overt DIC in various types of malignancy.

The extent of liver steatosis in chronic hepatitis C virus infection is mirrored by caspase activity in serum

Hepatic steatosis is a frequent histological alteration in chronic hepatitis C virus (HCV) infection that sensitizes the liver to cell injury, inflammation, and fibrosis via unclear mechanisms. Although apoptosis has been implicated in various liver diseases, its importance in HCV‐associated steatosis is largely unknown. In this study, we investigated the role of caspases, the key regulators of apoptosis, and employed two novel caspase assays, an immunological and a luminometric enzyme test, to detect hepatic caspase activation in sera from HCV patients with different grades of steatosis. Our data show that increased caspase activation can be found not only in liver biopsies, but also in sera from HCV patients with liver steatosis. Patients with steatosis exhibited significantly higher serum levels of caspase activity compared with normal healthy individuals. Moreover, the extent of steatosis closely correlated with serum caspase activity, whereas in particular in cases of low or moderate steatosis, no correlation was found with aminotransferase levels. In conclusion, apoptotic caspase activation is considerably elevated in HCV‐associated steatosis. More importantly, our data imply that measurement of caspase activation might be a sensitive serum biomarker to detect liver steatosis in patients with chronic HCV infection and other liver diseases. (HEPATOLOGY 2005.)

Tissue factor procoagulant activity of plasma microparticles is increased in patients with early-stage prostate cancer

Tissue factor (TF) plays a critical role in tumour growth and metastasis, and its enhanced release into plasma in association with cellular microparticles (MPs) has recently been associated with pathological cancer progression. We have previously demonstrated significantly elevated levels of plasma TF antigen as well as systemic coagulation and platelet activation in patients with localised prostate cancer. In this prospective study, we used a highly sensitive one-stage clotting assay to measure preoperative TF-specific procoagulant activity (PCA) of plasma MPs in 68 consecutive patients with early-stage prostate cancer to further explore the relevance of circulating TF in this tumour entity. Automated calibrated thrombography was used to monitor thrombin generation in cell-free plasma samples in the absence of exogenous TF or phospholipids. Compared to healthy male controls (n=20), patients had significantly increased levels of both D-dimer and TF-specific PCA of plasma MPs (p<0.001). Furthermore, MP-associated TF PCA was higher in patients with (n=29) than in those without (n=39) laboratory evidence of an acute-phase reaction (p=0.004) and decreased to normal levels within one week after radical prostatectomy. Overall, we found a significant correlation between TF-specific PCA of plasma MPs and plasma D-dimer (p=0.002), suggesting that plasma MPs contributed to in-vivo coagulation activation in a TF-dependent manner. Thrombin generation in plasma was also significantly increased in patients compared to controls (p<0.01). Collectively, our findings suggest that TF-specific PCA of plasma MPs contributes to intravascular coagulation activation in patients with early-stage prostate cancer and may represent a potential link between hypercoagulability, inflammation, and disease progression.

Undifferentiated embryonal sarcoma of the liver in adults

Undifferentiated embryonal sarcoma of the liver (UESL), a rare tumor that predominantly affects children, generally has been considered an aggressive neoplasm with an unfavorable prognosis. More recent reports have indicated that modern multimodal treatment and supportive care improve the survival of children with UESL. Data regarding the treatment and survival of adults have not been reviewed comprehensively, and only a few adult patients with UESL have been reported in the literature.

Rapid activation of monocyte tissue factor by antithymocyte globulin is dependent on complement and protein disulfide isomerase

Lymphocyte depletion with antithymocyte globulin (ATG) can be complicated by systemic coagulation activation. We found that ATG activated tissue factor procoagulant activity (TF PCA) on monocytic cells more potently than other stimuli that decrypt TF, including cell disruption, TF pathway inhibitor inhibition, and calcium ionophore treatment. Induction of TF PCA by ATG was dependent on lipid raft integrity and complement activation. We showed that ATG-mediated TF activation required complement activation until assembly of the C5b-7 membrane insertion complex, but not lytic pore formation by the membrane attack complex C5b-9. Consistently, induction of TF PCA by ATG did not require maximal phosphatidylserine membrane exposure and was not correlated with the magnitude of complement-induced lytic cell injury. Blockade of free thiols, an inhibitory monoclonal antibody to protein disulfide isomerase (PDI), and the small-molecule PDI antagonist quercetin-3-rutinoside prevented ATG-mediated TF activation, and C5 complement activation resulted in oxidation of cell surface PDI. This rapid and potent mechanism of cellular TF activation represents a novel connection between the complement system and cell surface PDI-mediated thiol-disulfide exchange. Delineation of this clinically relevant mechanism of activation of the extrinsic coagulation pathway during immunosuppressive therapy with ATG may have broader implications for vascular thrombosis associated with inflammatory disorders.

Important Role for CC Chemokine Ligand 2-Dependent Lung Mononuclear Phagocyte Recruitment to Inhibit Sepsis in Mice Infected with Streptococcus pneumoniae

The monocyte chemoattractant CCL2 is of major importance in inflammatory monocyte recruitment to the lungs in response to bacterial infection. Streptococcus pneumoniae is the most prevalent pathogen in community-acquired pneumonia causing significant morbidity and mortality worldwide. In the current study, we examined the role of CCL2 in lung-protective immunity against two strains of S. pneumoniae exhibiting different virulence profiles. Both wild-type mice and CCL2 knockout (KO) mice became septic within 24 h of infection with serotype 3 S. pneumoniae and died of infection by day 4 after challenge. In contrast, wild-type mice challenged with serotype 19 S. pneumoniae did not become septic or succumb to pneumococcal pneumonia, whereas CCL2 KO mice showed an early bacterial outgrowth in their lungs and sepsis starting by day 2 after infection, finally resulting in ∼50% decreased survival compared with wild-type mice. This phenotype was not due to impaired lung neutrophil recruitment in the KO mice, but was characterized by a significantly reduced recruitment of lung exudate macrophages and conventional lung dendritic cells, suggesting that these two phagocyte subsets critically regulate protection against septic disease progression in mice. In conclusion, we show here a differential role for CCL2-dependent lung exudate macrophage and conventional dendritic cell recruitment that critically contributes to lung protective immunity against S. pneumoniae.