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Complement protease MASP-1 activates human endothelial cells: PAR4 activation is a link between complement and endothelial function
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Abstracts / Molecular Immunology 46 (2009) 2818–2871
revealed the role of complement alternative pathway (AP), since factor B-deficient mice were protected from the disease. Our aim was to investigate the ability of neutrophil (PMN) to activate complement and a possible modulation by ANCAs. Whole blood incubation, with lepirudin (thrombin inhibitor) as anticoagulant, resulted in cell-bound C3d on PMN and monocytes, as measured by flow cytometry. C3c and iC3b were also present. C3 activation on phagocytes, resulting presumably from clotting factors upstream of thrombin, was enhanced by leukocyte activation by TNF/fMLP or by PMA, while lymphocytes remained devoid of C3d. In whole blood in EGTA/Mg, which blocked coagulation while allowing the complement alternative pathway, phagocyte-bound C3 was observed exclusively when TNF/fMLP or PMA were added. Similarly, TNF-activated isolated PMN, when incubated in serum, triggered the AP, as shown by C3d deposits in EGTA/Mg or C2depleted, but not in factor B-depleted serum. Cytokine-activated PMN released properdin (P), measured with a specific ELISA. Moreover, large amounts of membrane associated P, able to stabilize membrane-bound C3/C5-convertase, were observed on activated PMN incubated in plasma. C3d levels on fluid phase TNF-activated PMN were amplified by the presence of anti-PMN (anti-CD44, HLA-A/B) mAbs, via classical pathway triggering, while anti-PR3 or anti-MPO mAbs had no effect. By contrast, anti-MPO and anti-PR3 mAbs enhanced C3d deposition on TNF-activated adherent PMN incubated in lepirudin-autologous plasma. In conclusion, cytokineactivated PMN trigger the AP, resulting in C3 deposits, which are enhanced by ANCAs after TNF-induced PMN adhesion, a condition known to allow the access of ANCAs to their antigens. doi:10.1016/j.molimm.2009.05.213 O28 Human anti-citrullinated protein antibodies (ACPA) not only activate the classical- but also the alternative complement pathway L.A. Trouw ∗ , E.M. Haisma, E.W. Levarht, D. van der Woude, A. IoanFacsinay, M.R. Daha, T.W. Huizinga, R.E. Toes Department of Rheumatology, Leiden University Medical Center, Leiden, The Netherlands Introduction: Recent studies in mice show that arthritis-inducing antibodies do not require the classical pathway of complement activation, the pathway traditionally associated with antibody mediated defenses. Surprisingly, they essentially employ the alternative pathway. In humans anti-citrullinated protein antibodies (ACPA) are prime suspects in the pathogenesis of rheumatoid arthritis. These antibodies recognize posttranslationally modified (citrullinated) proteins that can be present in the joint. Here we have analyzed if ACPA activate complement and which pathways of complement are activated by ACPA. Methods: We have set up assays to measure complement activation by these antibodies by using cyclic citrullinated peptide (CCP2) coated plates, specific buffers that allow binding of ACPA without inducing complement activation during the binding step, followed by complement activation from a pool of normal human serum. Specific activation of different complement pathways was achieved by using specific buffers and C1q or MBL deficient sera. ACPA isotype usage was tested in relation to complement activation. Results: We observed that ACPA activate complement in a dose dependent manner via the classical and intriguingly also strongly via the alternative pathway, mimicking the situation in mice. We did not observe any lectin pathway activation, also not in those patients positive for IgA or IgM ACPA. Complement activation proceeded in vitro up to the formation of the membrane attack complex C5b9, showing that all activation
steps, including the release of C5a, have been taken. The extent of complement activation is proportional to the ACPA titer. Conclusions: These data show that ACPA not only activate the classical pathway but also strongly activate the alternative pathway. These findings provide a rationale to target the alternative pathway to inhibit disease progression in RA patients. doi:10.1016/j.molimm.2009.05.214 Session 5: Complement activation, regulation, adaptive immune response. 7 September, 9:10–10:40 O29 Contributions of MASP-1 and MASP-3 to fat metabolism by activation of complement factor D Minoru Takahashi ∗ , Daisuke Iwaki, Yuichi Endo, Teizo Fujita Department of Immunology and Fukushima Medical University School of Medicine, Fukushima 960-1295, Japan Complement factor D (Df), which is a key enzyme of the alternative pathway, constitutively exists in an active form in the circulation. Adipocytes mainly produce and secrete Df into peripheral blood. Several experimental data revealed that the activation peptide of pro-factor D (pro-Df) may be removed by the putative trypsin-like protease during the secretion in adipocytes, but the physiological activator of pro-Df was unknown. We found that MASP-1 and MASP-3, known as serine protease for the lectin pathway, contribute to activate Df by cleaving activation peptide in pro-Df, since almost Df in sera of Masp1/3-double knockout (Masp1/3−/− ) mouse still exists as pro-Df. No alterative pathway activation was observed in their sera. It was suggested that alternative pathway activation influences fat storage in adipocytes. Masp1/3−/− mice are also apparently lean, strongly indicating the contribution of MASP-1 and MASP-3 to fat metabolism. Here we report that parametrial adipose tissue weights were significantly lower in Masp1/3−/− mice to compare with those in wild-type mice, although other adipose tissues such as pararenal and subcutaneous adipose tissues did not show difference significantly. Level of leptin in female Masp1/3−/− mice reduced to compare that in wild-type mouse. In conclusion, abnormality of pro-Df activation in Masp1/3−/− mice was involved in the especial development of parametrial adipose tissues. doi:10.1016/j.molimm.2009.05.215 O30 Complement protease MASP-1 activates human endothelial cells: PAR4 activation is a link between complement and endothelial function Márton Megyeri a,∗ , Veronika Makó b , László Beinrohr a , Zoltán Doleschall c , Zoltán Prohászka b , László Cervenak b , Péter Závodszky a , Péter Gál a a
Institute of Enzymology, Biological Research Center, Hungarian Academy of Sciences, Budapest, Hungary b Semmelweis University, 3rd Department of Medicine, Budapest, Hungary c National Institute of Oncology, Department of Pathogenetics, Budapest, Hungary Activation of the complement system can induce and enhance inflammatory reaction. Mannose-binding lectin-associated serine protease-1 (MASP-1) is an abundant protease of the lectin pathway of complement. Several protein substrates have been reported
Abstracts / Molecular Immunology 46 (2009) 2818–2871
for MASP-1, however, its physiological function is debated since its discovery. We demonstrate for the first time that MASP-1 is able to activate Ca2+ -signaling, NFB and p38 MAPK pathways in cultured, human umbilical vein endothelial cells (HUVECs). Endothelial cells influence leukocyte function and control the inflammatory reaction by changing vascular permeability, perfusion and coagulation. The activation of endothelial cells was initiated by MASP-1 only, the related protease – MASP-2 – had no such effect. The phenomenon was dependent on the proteolytic activity of MASP1, suggesting modulation of endothelial cell function through a protease-activated receptor (PAR). Using synthetic peptide substrates representing the protease-sensitive regions of PARs, we were able to demonstrate that PAR4 is a target of MASP-1. The presence of functionally active PAR4 in HUVECs was proven using PAR4 agonist peptide and mRNA quantification. Finally, we showed that the amount of membrane-bound intact PAR4 decreases after MASP-1 treatment. All these results provide a novel link between the regulation of endothelial cell function and complement system activation, and suggest that MASP-1 induced PAR4 activation could contribute to the development of the inflammatory reaction. doi:10.1016/j.molimm.2009.05.216 O31 Cartilage oligomeric matrix protein (COMP) has a dual role in complement regulation K.E. Happonen a,∗ , A. Pramhed b , D. Heinegård b , A.M. Blom a a
Department of Laboratory Medicine, Wallenberg Laboratory, University Hospital Malmö, Lund University, Sweden b Department of Experimental Medical Science, BMC, Lund University, Sweden Cartilage oligomeric matrix protein (COMP) is a structural component of cartilage where it serves as a catalyst of collagen fibrillogenesis. In addition, a wider expression pattern in other extracellular matrices has been reported. Elevated levels of COMP can be found in serum during degeneration of cartilage associated with active joint disease, as well as in active stage of systemic sclerosis with skin involvement. Since some cartilage proteins have previously been shown to regulate complement activation, we sought to determine whether COMP interacts with the complement system as well. Here we show that COMP inhibits the classical pathway of complement in both haemolytic assay and complement deposition assay and that this inhibition is due to direct interaction with the stalk-region of C1q. Furthermore, we found that COMP induces deposition of C9 via the alternative pathway, which is attributable to a direct interaction with C3. Both the intact pentameric protein and single monomeric chains showed these complement regulatory properties. In addition, we determined the binding region for both C1q and C3 in COMP. To our knowledge COMP is the first cartilage protein that activates one complement pathway and simultaneously inhibits another. The net outcome of these interactions is most likely determined by the levels of both free COMP and available complement proteins. The ability of COMP to regulate the complement system might be of importance during states where cartilage is abnormally eroded such as during osteoarthritis or knee injuries. doi:10.1016/j.molimm.2009.05.217
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O32 Physiological upregulation of CR1 and Fc␥RII on memory B cells is lacking in SLE patients, but is not related to the cells’ activation state Anna Erdei a,c,∗ , Andrea Isaák a , Mariann Kremlitzka a , Gyula Poór b a
Department of Immunology, Eötvös Loránd University, Budapest, Hungary b Institute of Rheumatology and Physiotherapy, Budapest, Hungary c Research Group of the Hungarian Academy of Sciences, Budapest, Hungary Under physiological conditions immune complexes (IC) are efficiently cleared from the circulation and meanwhile provide important feedback signals for the immune system via Fc␥Rs and complement receptors. These mechanisms might be defected in certain autoimmune diseases, which are often characterized by the appearance of elevated levels of self-reactive antibodies, indicating also the dysregulation of the B-cell compartment. Perturbations in the expression of the IC-binding Fc␥-and complement receptors that modulate the BCR-mediated signal can lead to aberrant activation and/or survival of autoreactive B cells, thus influencing the B-cell compartment. To clarify which B-cell subsets are affected in SLE, we investigated the expression of the IC-binding receptors on various B-cell populations. We found that in SLE there is a disturbed ratio of naïve and memory B cells (53% versus 47%) as compared to healthy individuals (75% versus 25%). In active SLE we detected a large expansion of CD27high plasmablast/early plasma cells, reaching 20 times the amount found in healthy donors. Investigating the expression of Fc␥RII (CD32), complement receptors CR1 and CR2 (CD21, CD35), and mIgG/IgM on various B-cell subpopulations we found reduced expression of CR1 and Fc␥RII on CD27+ memory B cells from SLE patients compared to healthy controls. The terminally differentiated CD19+ /CD27high /sIglow plasmablast population displayed very low levels of Fc␥RII and CR1, both in the diseased and control individuals. We also clarified that the expression pattern of CR1 in peripheral B-cell subpopulations resembles that of Fc␥RII but not of CR2 both under physiological conditions, and in SLE. As we found that activation results in a strongly reduced expression of CR1 and Fc␥RII on tonsillar memory B cell, we set out to investigate the influence of activation also on peripheral B cells. In contrast to tonsillar B cells the expression levels of CR1 and Fc␥RII were similar both on resting and activated cells in the memory compartment. Based on these results we conclude that the decreased expression of CR1 and Fc␥RII on the memory B cells in SLE is not related to their activation status. doi:10.1016/j.molimm.2009.05.218 O33 CD46-mediated Treg induction requires intracellular CD46 processing by gamma-secretase Gaëlle Le Friec King’s College London, United Kingdom The concurrent activation of the T cell receptor and CD46 on human CD4+ T cells induces adaptive IL-10-secreting regulatory T cells (Treg). CD46 is expressed in four isoforms with two distinct, alternatively spliced cytoplasmic tails, CYT-1 or CYT-2. Both tails can transmit intracellular signals upon CD46 crosslinking but their single contributions to Treg development are unknown. Also, CD46 is downregulated after its activation on most cells either via cleavage by metalloproteinases or by internalization, the biological
Abstracts / Molecular Immunology 46 (2009) 2818–2871
revealed the role of complement alternative pathway (AP), since factor B-deficient mice were protected from the disease. Our aim was to investigate the ability of neutrophil (PMN) to activate complement and a possible modulation by ANCAs. Whole blood incubation, with lepirudin (thrombin inhibitor) as anticoagulant, resulted in cell-bound C3d on PMN and monocytes, as measured by flow cytometry. C3c and iC3b were also present. C3 activation on phagocytes, resulting presumably from clotting factors upstream of thrombin, was enhanced by leukocyte activation by TNF/fMLP or by PMA, while lymphocytes remained devoid of C3d. In whole blood in EGTA/Mg, which blocked coagulation while allowing the complement alternative pathway, phagocyte-bound C3 was observed exclusively when TNF/fMLP or PMA were added. Similarly, TNF-activated isolated PMN, when incubated in serum, triggered the AP, as shown by C3d deposits in EGTA/Mg or C2depleted, but not in factor B-depleted serum. Cytokine-activated PMN released properdin (P), measured with a specific ELISA. Moreover, large amounts of membrane associated P, able to stabilize membrane-bound C3/C5-convertase, were observed on activated PMN incubated in plasma. C3d levels on fluid phase TNF-activated PMN were amplified by the presence of anti-PMN (anti-CD44, HLA-A/B) mAbs, via classical pathway triggering, while anti-PR3 or anti-MPO mAbs had no effect. By contrast, anti-MPO and anti-PR3 mAbs enhanced C3d deposition on TNF-activated adherent PMN incubated in lepirudin-autologous plasma. In conclusion, cytokineactivated PMN trigger the AP, resulting in C3 deposits, which are enhanced by ANCAs after TNF-induced PMN adhesion, a condition known to allow the access of ANCAs to their antigens. doi:10.1016/j.molimm.2009.05.213 O28 Human anti-citrullinated protein antibodies (ACPA) not only activate the classical- but also the alternative complement pathway L.A. Trouw ∗ , E.M. Haisma, E.W. Levarht, D. van der Woude, A. IoanFacsinay, M.R. Daha, T.W. Huizinga, R.E. Toes Department of Rheumatology, Leiden University Medical Center, Leiden, The Netherlands Introduction: Recent studies in mice show that arthritis-inducing antibodies do not require the classical pathway of complement activation, the pathway traditionally associated with antibody mediated defenses. Surprisingly, they essentially employ the alternative pathway. In humans anti-citrullinated protein antibodies (ACPA) are prime suspects in the pathogenesis of rheumatoid arthritis. These antibodies recognize posttranslationally modified (citrullinated) proteins that can be present in the joint. Here we have analyzed if ACPA activate complement and which pathways of complement are activated by ACPA. Methods: We have set up assays to measure complement activation by these antibodies by using cyclic citrullinated peptide (CCP2) coated plates, specific buffers that allow binding of ACPA without inducing complement activation during the binding step, followed by complement activation from a pool of normal human serum. Specific activation of different complement pathways was achieved by using specific buffers and C1q or MBL deficient sera. ACPA isotype usage was tested in relation to complement activation. Results: We observed that ACPA activate complement in a dose dependent manner via the classical and intriguingly also strongly via the alternative pathway, mimicking the situation in mice. We did not observe any lectin pathway activation, also not in those patients positive for IgA or IgM ACPA. Complement activation proceeded in vitro up to the formation of the membrane attack complex C5b9, showing that all activation
steps, including the release of C5a, have been taken. The extent of complement activation is proportional to the ACPA titer. Conclusions: These data show that ACPA not only activate the classical pathway but also strongly activate the alternative pathway. These findings provide a rationale to target the alternative pathway to inhibit disease progression in RA patients. doi:10.1016/j.molimm.2009.05.214 Session 5: Complement activation, regulation, adaptive immune response. 7 September, 9:10–10:40 O29 Contributions of MASP-1 and MASP-3 to fat metabolism by activation of complement factor D Minoru Takahashi ∗ , Daisuke Iwaki, Yuichi Endo, Teizo Fujita Department of Immunology and Fukushima Medical University School of Medicine, Fukushima 960-1295, Japan Complement factor D (Df), which is a key enzyme of the alternative pathway, constitutively exists in an active form in the circulation. Adipocytes mainly produce and secrete Df into peripheral blood. Several experimental data revealed that the activation peptide of pro-factor D (pro-Df) may be removed by the putative trypsin-like protease during the secretion in adipocytes, but the physiological activator of pro-Df was unknown. We found that MASP-1 and MASP-3, known as serine protease for the lectin pathway, contribute to activate Df by cleaving activation peptide in pro-Df, since almost Df in sera of Masp1/3-double knockout (Masp1/3−/− ) mouse still exists as pro-Df. No alterative pathway activation was observed in their sera. It was suggested that alternative pathway activation influences fat storage in adipocytes. Masp1/3−/− mice are also apparently lean, strongly indicating the contribution of MASP-1 and MASP-3 to fat metabolism. Here we report that parametrial adipose tissue weights were significantly lower in Masp1/3−/− mice to compare with those in wild-type mice, although other adipose tissues such as pararenal and subcutaneous adipose tissues did not show difference significantly. Level of leptin in female Masp1/3−/− mice reduced to compare that in wild-type mouse. In conclusion, abnormality of pro-Df activation in Masp1/3−/− mice was involved in the especial development of parametrial adipose tissues. doi:10.1016/j.molimm.2009.05.215 O30 Complement protease MASP-1 activates human endothelial cells: PAR4 activation is a link between complement and endothelial function Márton Megyeri a,∗ , Veronika Makó b , László Beinrohr a , Zoltán Doleschall c , Zoltán Prohászka b , László Cervenak b , Péter Závodszky a , Péter Gál a a
Institute of Enzymology, Biological Research Center, Hungarian Academy of Sciences, Budapest, Hungary b Semmelweis University, 3rd Department of Medicine, Budapest, Hungary c National Institute of Oncology, Department of Pathogenetics, Budapest, Hungary Activation of the complement system can induce and enhance inflammatory reaction. Mannose-binding lectin-associated serine protease-1 (MASP-1) is an abundant protease of the lectin pathway of complement. Several protein substrates have been reported
Abstracts / Molecular Immunology 46 (2009) 2818–2871
for MASP-1, however, its physiological function is debated since its discovery. We demonstrate for the first time that MASP-1 is able to activate Ca2+ -signaling, NFB and p38 MAPK pathways in cultured, human umbilical vein endothelial cells (HUVECs). Endothelial cells influence leukocyte function and control the inflammatory reaction by changing vascular permeability, perfusion and coagulation. The activation of endothelial cells was initiated by MASP-1 only, the related protease – MASP-2 – had no such effect. The phenomenon was dependent on the proteolytic activity of MASP1, suggesting modulation of endothelial cell function through a protease-activated receptor (PAR). Using synthetic peptide substrates representing the protease-sensitive regions of PARs, we were able to demonstrate that PAR4 is a target of MASP-1. The presence of functionally active PAR4 in HUVECs was proven using PAR4 agonist peptide and mRNA quantification. Finally, we showed that the amount of membrane-bound intact PAR4 decreases after MASP-1 treatment. All these results provide a novel link between the regulation of endothelial cell function and complement system activation, and suggest that MASP-1 induced PAR4 activation could contribute to the development of the inflammatory reaction. doi:10.1016/j.molimm.2009.05.216 O31 Cartilage oligomeric matrix protein (COMP) has a dual role in complement regulation K.E. Happonen a,∗ , A. Pramhed b , D. Heinegård b , A.M. Blom a a
Department of Laboratory Medicine, Wallenberg Laboratory, University Hospital Malmö, Lund University, Sweden b Department of Experimental Medical Science, BMC, Lund University, Sweden Cartilage oligomeric matrix protein (COMP) is a structural component of cartilage where it serves as a catalyst of collagen fibrillogenesis. In addition, a wider expression pattern in other extracellular matrices has been reported. Elevated levels of COMP can be found in serum during degeneration of cartilage associated with active joint disease, as well as in active stage of systemic sclerosis with skin involvement. Since some cartilage proteins have previously been shown to regulate complement activation, we sought to determine whether COMP interacts with the complement system as well. Here we show that COMP inhibits the classical pathway of complement in both haemolytic assay and complement deposition assay and that this inhibition is due to direct interaction with the stalk-region of C1q. Furthermore, we found that COMP induces deposition of C9 via the alternative pathway, which is attributable to a direct interaction with C3. Both the intact pentameric protein and single monomeric chains showed these complement regulatory properties. In addition, we determined the binding region for both C1q and C3 in COMP. To our knowledge COMP is the first cartilage protein that activates one complement pathway and simultaneously inhibits another. The net outcome of these interactions is most likely determined by the levels of both free COMP and available complement proteins. The ability of COMP to regulate the complement system might be of importance during states where cartilage is abnormally eroded such as during osteoarthritis or knee injuries. doi:10.1016/j.molimm.2009.05.217
2829
O32 Physiological upregulation of CR1 and Fc␥RII on memory B cells is lacking in SLE patients, but is not related to the cells’ activation state Anna Erdei a,c,∗ , Andrea Isaák a , Mariann Kremlitzka a , Gyula Poór b a
Department of Immunology, Eötvös Loránd University, Budapest, Hungary b Institute of Rheumatology and Physiotherapy, Budapest, Hungary c Research Group of the Hungarian Academy of Sciences, Budapest, Hungary Under physiological conditions immune complexes (IC) are efficiently cleared from the circulation and meanwhile provide important feedback signals for the immune system via Fc␥Rs and complement receptors. These mechanisms might be defected in certain autoimmune diseases, which are often characterized by the appearance of elevated levels of self-reactive antibodies, indicating also the dysregulation of the B-cell compartment. Perturbations in the expression of the IC-binding Fc␥-and complement receptors that modulate the BCR-mediated signal can lead to aberrant activation and/or survival of autoreactive B cells, thus influencing the B-cell compartment. To clarify which B-cell subsets are affected in SLE, we investigated the expression of the IC-binding receptors on various B-cell populations. We found that in SLE there is a disturbed ratio of naïve and memory B cells (53% versus 47%) as compared to healthy individuals (75% versus 25%). In active SLE we detected a large expansion of CD27high plasmablast/early plasma cells, reaching 20 times the amount found in healthy donors. Investigating the expression of Fc␥RII (CD32), complement receptors CR1 and CR2 (CD21, CD35), and mIgG/IgM on various B-cell subpopulations we found reduced expression of CR1 and Fc␥RII on CD27+ memory B cells from SLE patients compared to healthy controls. The terminally differentiated CD19+ /CD27high /sIglow plasmablast population displayed very low levels of Fc␥RII and CR1, both in the diseased and control individuals. We also clarified that the expression pattern of CR1 in peripheral B-cell subpopulations resembles that of Fc␥RII but not of CR2 both under physiological conditions, and in SLE. As we found that activation results in a strongly reduced expression of CR1 and Fc␥RII on tonsillar memory B cell, we set out to investigate the influence of activation also on peripheral B cells. In contrast to tonsillar B cells the expression levels of CR1 and Fc␥RII were similar both on resting and activated cells in the memory compartment. Based on these results we conclude that the decreased expression of CR1 and Fc␥RII on the memory B cells in SLE is not related to their activation status. doi:10.1016/j.molimm.2009.05.218 O33 CD46-mediated Treg induction requires intracellular CD46 processing by gamma-secretase Gaëlle Le Friec King’s College London, United Kingdom The concurrent activation of the T cell receptor and CD46 on human CD4+ T cells induces adaptive IL-10-secreting regulatory T cells (Treg). CD46 is expressed in four isoforms with two distinct, alternatively spliced cytoplasmic tails, CYT-1 or CYT-2. Both tails can transmit intracellular signals upon CD46 crosslinking but their single contributions to Treg development are unknown. Also, CD46 is downregulated after its activation on most cells either via cleavage by metalloproteinases or by internalization, the biological