Michael Loos

Intracoronary Application of C1 Esterase Inhibitor Improves Cardiac Function and Reduces Myocardial Necrosis in an Experimental Model of Ischemia and Reperfusion

BACKGROUND Myocardial injury from ischemia can be aggravated by reperfusion of the jeopardized area. The precise underlying mechanisms have not been clearly defined, but proinflammatory events, including complement activation, leukocyte adhesion, and infiltration and release of diverse mediators, probably play important roles. The present study addresses the possibility of reducing reperfusion damage by the application of C1 esterase inhibitor (C1-INH). METHODS AND RESULTS Cardioprotection by C1-INH 20 IU/kg IC was examined in a pig model with 60 minutes of coronary occlusion, followed by 120 minutes of reperfusion. C1-INH was administered during the first 5 minutes of coronary reperfusion Compared with the NaCl controls, C1-INH reduced myocardial injury (48.8 +/- 7.8% versus 73.4 +/- 4.0% necrosis of area at risk, P < or = .018). C1-INH treatment significantly reduced circulating C3a and slightly attenuated C5a plasma concentrations. Myocardial protection was accompanied by reduced plasma concentration of creatine kinase and troponin-T. C1-INH had no effect on global hemodynamic parameters, but local myocardial contractility was markedly improved in the ischemic zone. In the short-axis view, 137 degrees of the anteroseptal region showed significantly improved wall motion at early and 29 degrees at late reperfusion with C1-INH treatment. CONCLUSIONS C1-INH significantly protects ischemic tissue from reperfusion damage, reduces myocardial necrosis, and improves local cardiac function.

Complement C1q Is Dramatically Up-Regulated in Brain Microglia in Response to Transient Global Cerebral Ischemia

Recent evidence suggests that the pathophysiology of neurodegenerative and inflammatory neurological diseases has a neuroimmunological component involving complement, an innate humoral immune defense system. The present study demonstrates the effects of experimentally induced global ischemia on the biosynthesis of C1q, the recognition subcomponent of the classical complement activation pathway, in the CNS. Using semiquantitative in situ hybridization, immunohistochemistry, and confocal laser scanning microscopy, a dramatic and widespread increase of C1q biosynthesis in rat brain microglia (but not in astrocytes or neurons) within 24 h after the ischemic insult was observed. A marked increase of C1q functional activity in cerebrospinal fluid taken 1, 24, and 72 h after the ischemic insult was determined by C1q-dependent hemolytic assay. In the light of the well-established role of complement and complement activation products in the initiation and maintenance of inflammation, the ischemia-induced increase of cerebral C1q biosynthesis and of C1q functional activity in the cerebrospinal fluid implies that the proinflammatory activities of locally produced complement are likely to contribute to the pathophysiology of cerebral ischemia. Pharmacological modulation of complement activation in the brain may be a therapeutic target in the treatment of stroke.

Expression of C1q, a subcomponent of the rat complement system, is dramatically enhanced in brains of rats with either Borna disease or experimental allergic encephalomyelitis

In situ hybridization, RT-PCR and Northern blot analysis as well immunohistochemistry were used to examine the expression of C1q, a subcomponent of the rat complement system, in brains of rats infected with Borna disease virus (BDV) and rats afflicted with experimental allergic encephalomyelitis (EAE) induced by the adoptive transfer of myelin basic protein specific T cells. C1q mRNA, which was not detected in normal brain, became clearly detectable using RT-PCR analysis by d14 post infection (p.i.) with BDV. Maximal levels of C1q mRNA were reached 21 days p.i. when inflammatory reactions in the brain were also at a peak. Similarly, C1q mRNA was elevated when the clinical symptoms of EAE became evident 5 days following cell transfer. C1q positive cells, as identified by immunohistology, were preferentially localized in grey and white matter of the hippocampus and basolateral cortex. The C1q positive cells resembled microglial cells in morphology. The correlation of C1q expression with the development of neurological disease as well as the dramatic increase of C1q within brain regions with inflammatory lesions suggest that local biosynthesis of C1q may play a role in the pathogenesis of Borna virus induced and autoimmune encephalomyelitis.

Mode of interaction of different polyanions with the first (C1, C1) the second (C2) and the fourth (C4) component of complement—I: Effect on fluid phase C1 and on C1 bound toEA or to EAC4☆☆☆

Abstract The effect of eight different polyanions was tested on fluid phase and on bound C 1 ( EAC 1 and EAC 1 4 ) as well as on purified C4 and C2. The polyanions had no effect on purified C4 and C2 but they showed a strong inhibition of C 1 activity. On the weight basis, 0·020 μg/ml of dextran sulfate, 0·020 μg/ml of polyvinyl sulfate, 0·030 μg/ml polyanethol sulfonate (liquoid) and 0·15 μg/ml of heparin inhibited 50% of free C 1 , containing 1−1·15 C1-sites per cell. To get the same inhibition effect on free C 1 , 1·6 μg/ml of poly I, 4·8 μg/ml of the pentosan polysulfoester Sp 54, 24 μg/ml of germanin and 68 μg/ml of chondroitin sulfate were necessary. Binding of C 1 to EA or EAC4 markedly reduced the effect of liquoid (100-fold), of dextran sulfate (90-fold), of heparin (47-fold) and of polyvinyl sulfate (40-fold) on C 1 ; in contrast, the effect of poly I, germanin, Sp 54 and chondroitin sulfate were reduced 6·3, 4·6 2·3 and 0·9-fold, respectively. The inhibition of C 1 was shown to be the result of a direct interaction of at least liquoid, dextran sulfate, polyvinyl sulfate and heparin with the C1 subunit Clq: preincubation of these substances with purified Clq reduced their inhibitory capacity against free C 1 . In addition, all polyanions testes reduced the consumption of C4 and C2 by C1, although there was no evidence for a direct interaction of polyanions with the isolated C1-esterase, C1s.

Differential Expression of the Murine Mannose-Binding Lectins A and C in Lymphoid and Nonlymphoid Organs and Tissues

Mannose-binding lectin (MBL), a member of the collectin family, binds to carbohydrate structures on the surfaces of micro-organisms and may serve as a recognition molecule of the lectin pathway of complement activation. In rodents two forms, MBL-A and MBL-C, were described and shown to be products of two related, but uncoupled, genes. The liver is the main source of MBL biosynthesis. For rat MBL-A, expression has also been described in the kidney. Here we report that the two forms of murine MBL are differentially expressed in a number of nonhepatic tissues. Real-time RT-PCR revealed that the liver is the major site of expression for both MBL genes. Lower copy numbers were found in kidney, brain, spleen, and muscle. In testis, only the MBL-A gene is expressed, whereas MBL-C is exclusively expressed in small intestine. Using in situ hybridization and immunohistochemistry, we demonstrate that both MBLs are synthesized by hepatocytes and show MBL expression in cells of the monocyte/macrophage lineage. In the kidney MBL-A, but not MBL-C, was found to be synthesized. Vice versa, only MBL-C biosynthesis was detected in endothelial cells of the small intestine. The latter finding may support the view that MBL-C, as part of the innate immune system, may be a counterpart of secretory IgA of the acquired immune system in preventing, for example, microbial invasion and colonization. Our findings demonstrate that MBL-A and MBL-C are differentially expressed, implying distinct biological roles for both recognition molecules of the murine lectin pathway of complement.

Reconstitution of the Complement Function in C1q-Deficient (C1qa−/−) Mice with Wild-Type Bone Marrow Cells

Besides Ab-independent and Ab-dependent activation of the complement classical pathway in host defense, C1q plays a key role in the processing of immune complexes and in the clearance of apoptotic cells. In humans, C1q deficiency leads to systemic lupus erythematosus-like symptoms in over 90% of the cases, thus making this defect a strong disease susceptibility factor. Similarly, C1q-deficient mice (C1qa−/−) develop systemic lupus erythematosus-like symptoms, such as autoantibodies and glomerulonephritis. We have previously provided evidence that C1q is produced by cells of the monocyte-macrophage lineage. In this study, we have tested whether transplantation of bone marrow cells would be sufficient to reconstitute C1q levels in C1qa−/− mice. C1qa−/− mice received a single graft of 107 bone marrow cells from wild-type (wt) donors after irradiation doses of 6, 7, 8, or 9 Gy. Engraftment was monitored by a Y chromosome-specific PCR and a PCR that differentiated wt from C1qa−/− genotype. Serum levels of C1q Ag and C1 function increased rapidly in the recipient mice, and titers reached normal levels within 6 wk after bone marrow transplantation. In wt mice that received C1qa−/− bone marrow, serum levels of C1q decreased constantly over time and became C1q deficient within 55 wk. These data clearly demonstrate that bone marrow-derived cells are the source of serum C1q and are competent to reconstitute normal C1q serum levels in C1q-deficient mice. Therefore, stem cell transplantation could be a therapy for patients with hereditary C1q deficiency.

Biosynthesis of the collagen-like C1q molecule and its receptor functions for Fc and polyanionic molecules on macrophages.

At the beginning of the nineteenth century, knowledge of immunity was limited to a few practical methods based on empirical observations, e.g., the observation by Jenner in 1798 that inoculation with cowpox material induced an immunity to smallpox. The discoveries by Louis Pasteur and Robert Koch that microorganisms caused fermentations and were responsible for a number of infectious diseases, greatly advanced the concepts of susceptibility and immunity in a limited number of diseases. In the late nineteenth century, the complement system was discovered by Fodor(1887), Nuttall(1888), and Buchner (1889a, b) through studying the bactericidal action of blood serum. It was recognized that killing of bacteria in fresh serum required at least two different substances: a heat-stable (30 min, 56 °C) factor, today known to be the antibody specific for the particular microorganism; and a heat-labile factor, which was normally present in each serum. This factor was at first termed “alexine” (Buchner 1889a, b) and later designated “complement” (Pfeiffer and Issaeff 1894; Bordet 1896). On 18 May 1889, Buchner made the following Statement at a lecture in Munich: “Das Vorhandensein bakterienfeindlicher Wirkungen durch flussige Bestandthile der Korpersafte lasst die uberall nachweisbare Thatigkeit der Phagozyten als weniger ausschlaggebend erkennen” (The presence of bactericidal action in body fluids reveals the overall detectable activity of phagocytes as less decisive). This statement by the chief advocate of the humoral theory of resistance to microbial infections was directed against the cellular theory of immunity proposed by Metchnikoff. In his answer to Buchner’s critique, Metchnikoff (1889; English translation 1905) came to the conclusion, based on his own work, that “the postulates of this theory are often not in accord with the real facts,” and that the bactericidal effect of body fluids has nothing in common with the phenomenon of immunity („la propriete bactericide des humeurs ne correspond nullement aux phenomenes de l’immunite“).

Autoantibodies against C1q : view on clinical relevance and pathogenic roles

C. E. H. SIEGERT, M. D. KAZATCHKINE*, A. SJO¨HOLM§, R. WU¨RZNER†, M. LOOS‡ & M. R. DAHADepartment of Nephrology, Leiden University Medical Centre, Leiden, The Netherlands, *Institut National de la Sante´et de laRecherche Medicale, Hopital Broussais, Paris, France, † Institut fu¨r Hygiene, Leopold-Franzens-Universita¨t, Innsbruck, Austria,‡ Institute for Medical Microbiology and Hygiene, J-Gutenberg University, Mainz, Germany, and§Clinical Microbiology Laboratory,Lund University Hospital, Lund, Sweden(Accepted for publication 6 January 1999)INTRODUCTIONImmune responses to autoantigens are quite common andautoimmunity is considered to be a physiological part of theimmune system. Autoantibodies are an inherent property of theantibody repertoire of many healthy individuals and are thereforereferred to as natural autoantibodies [1,2]. The numerous kinds ofautoantibodies fall into two main categories, depending on whetherorgan-specific or non-organ-specific autoantigens are involved.Non-organ-specific antigens mostly occur in nucleated cells,such as DNA, or are found among circulating plasma proteins,such as coagulant proteins and the Fc portion of IgG.Natural autoantibodies have been proposed to be involved inthe clearance of degradation products that are formed during cellmetabolism. In this respect natural autoantibodies are also referredto as housekeeping antibodies [3]. Autoantibodies may also beassociated with disease states but do not necessarily play a role inthe pathogenesis of such diseases. The presence of autoantibodiesmay for example be secondary to the production of tissue damage,or independent pathogenic factors may directly induce both thedisease and the presence of autoantibodies.In 1984 autoantibodies to C1q (C1qAb) were reported to bepresent in serum of patients with systemic lupus erythematosus(SLE) [4]. The recognition that C1q may serve as a non-organ-specific autoantigen has attracted a growing number ofinvestigators. This study discusses the knowledge of C1q asautoantigen by reviewing the epidemiology, disease associations,and pathophysiology of C1qAb.ROLE OF C1q IN IMMUNE COMPLEXCLEARANCEActivation of the complement system is the first step in theprevention of damage by immune complexes. Initiation of com-plement activation occurs through three pathways: the classical,the alternative, and the lectin pathway. The classical pathway ofthe complement system is considered to be the most importantpathway in immune complex clearance. This pathway may beactivated by IgM- and IgG-containing immune complexes afterbinding of C1q [5]. C1q is a subcomponent of the first component(C1) of the classical pathway. It is a large highly cationicglycoprotein with a molecular weight of 410kD. C1q consists ofsix copies each of three polypeptide chains, A, B, and C. The A, B,and C chains are rich in hydroxylated amino acids and are linkedtogether by disulphide bonds into dimers [6] Together these dimersform a triple helix structure which resembles collagen. Towardsthe N-terminal end of C1q the triple helices lay parallel to eachother and towards the C-terminal end they diverge. The N-terminalend is called the collagen-like region which is linked by theconnecting strands to the C-terminal end, which is called theglobular heads region. The macromolecular structure of C1q issaid to resemble a bunch of tulips [5–7].The function of C1q is directly related to its structure. Bindingof Fc regions of immunoglobulins to the globular head portions ofC1q induces distortion of the connecting strand which changes theconformation of the collagen-like region [8,9]. The dynamicequilibrium between C1q and the other subcomponents of C1,C1r and C1s, subsequently shifts and induces further activation ofthe cascade of proteins composing the classical pathway. Thisresults in the prevention of lattice formation of immune complexesand ensures their clearance from the circulation by the mono-nuclear phagocyte system [10]. Although the recognition protein ofthe lectin pathway, mannose-binding lectin, is structurally relatedto C1q, it is not known to be involved in immune complexclearance mechanisms [11]. To summarize, activation of C1 bybinding of immune complexes to C1q is a prerequisite for immunecomplex clearance.HISTORY OF C1qAbSince SLE is considered to be the prototype of immune complexdiseases in man, a large variety of immune complex assays hasbeen employed to investigate possible pathogenic roles of circulat-ing immune complexes and to relate their titres to the presence ofmanifestations of the disease. The solid-phase C1q binding assay isone of the most frequently used assays for both purposes [12]. Thisradioimmunoassay is based on the binding of immune complexesto C1q, which is fixed to a solid phase. Studies in the early 1970s

Complete functional C1q deficiency associated with systemic lupus erythematosus (SLE).

A complete functional deficiency of Clq is described in a patient suffering from SLE. From reduced plasma C1 activity of the parents a hereditary trait was assumed. The defective C1q molecule was haemolytically inactive, did not bind to immune complexes, and was not recognized by the monocyte C1q receptor. C1 activity in the patients serum could be restored by the addition of purified C1q. Analysis by gelfiltration and ultracentrifugation experiments revealed an immunoreactive molecule of about 150 kD mol. wt, corresponding to one structural subunit of the C1q macromolccule, containing two A chain‐B chain dimers and a C‐C chain dimer. Applying Southern blot analysis with cDNA clones encoding for the three individual chains of the C1q molecule, no restriction fragment length polymorphism was detected, ruling out possible major alterations of the genetic information.

IL-17 regulates gene expression and protein synthesis of the complement system, C3 and factor B, in skin fibroblasts

Human IL‐17 is a cytokine secreted by CD4+‐activated memory T cells with the profile of effects of a Th1 cytokine. The effects of IL‐17 on many cellular constituents of joints suggest that it may participate in inflammatory joint diseases. Proteins of the complement system are known to be regulated by pro‐ and anti‐inflammatory cytokines. The purpose of this work was to study the effect of IL‐17 alone and combined with tumour necrosis factor (TNF) on the expression and synthesis of factor B and C3. Fibroblasts were stimulated with the relevant cytokine or cytokines, pulse labelled with 35S‐methionine, and the newly synthesized proteins were immunoprecipitated and subjected to SDS–PAGE. Gene expression was determined by Northern blot analysis. IL‐17 10 ng/ml induced increases in gene expression and protein synthesis of C3, 2·25 ± 0·26‐ and 2·7 ± 0·7‐fold, respectively, with concomitant non‐significant effects on factor B, 1·5 ± 0·45‐ and 2·2 ± 1·2‐fold, respectively. When both IL‐17 and TNF were present simultaneously, the synthesis of factor B increased by 85% more than the expected additive effects of these cytokines separately, while for C3 the effect of both cytokines was 19% lower than the expected additive effect (observed/expected = 0·81). IL‐4 reduced the synergistic effect by 50%. We conclude that IL‐17 has a regulatory role on C3 expression and synthesis and an amplifying effect on TNF‐induced factor B synthesis. Taken together with the evidence that TNF is a major cytokine involved in the inflammation of rheumatoid arthritis, it suggests that IL‐17 has a proinflammatory role in the inflammation process of joints. The distinct effects of IL‐4, IL‐17 and TNF on the synthesis of factor B in fibroblasts suggest that factor B and the alternative pathway of the complement system may play an important role in joint inflammation.