Complement Split Product Research Articles

Vascular deposition of complement-split products in kidney allografts with cell-mediated rejection

Complement activation in 73 renal transplant biopsies was investigated by indirect immunoperoxidase staining using MoAbs reactive with complement-split products. Intense deposition of complement fragments C4d and C3d in peritubular capillaries, indicating activation of the classical pathway, could be detected in the majority of transplanted kidneys with cell-mediated rejections. Abundant deposition of complement-split products was observed in 22 early biopsies from patients with high 'immunological risk' (i.e. previous, rejected transplants and/or circulating antibodies against HLA-antigens). Despite negative results in the crossmatch before transplantation and paucity of immunoglobulins in transplant biopsies, antibodies directed against endothelial cell antigens should be considered as a possible cause of classical complement activation.

Complement split products and proinflammatory cytokines in intraoperatively salvaged unwashed blood during hip replacement: comparison between heparin‐coated and non‐heparin‐coated autotransfusion systems

The aim of the present study was to investigate the quality of shed blood collected in a new intraoperative autotransfusion system (Sangvia, AstraTech, Sweden) and to study whether heparin-coated surfaces in the device reduce the production of inflammatory mediators. The study was randomized and prospective. Twelve total hip arthroplasty patients whose blood was collected with a device having a heparin-coated surface and 12 patients whose blood was collected with a device having a non-heparin-coated surface were included. Venous blood was drawn from the patients preoperatively. Intraoperatively 200 ml salvaged blood was collected and samples were also withdrawn; samples were obtained from the blood bag. Compared to venous blood, elevated concentrations of interleukin 6 (IL-6), IL-8, C3a and polymorphonuclear elastase were found in collected blood. No significant differences in inflammatory mediators were found between the heparin-coated and the non-heparin-coated groups. The median haemoglobin concentration in the salvaged blood was 74 g/l in both groups. Plasma haemoglobin and potassium concentrations were also elevated. There were no significant differences between the groups. The present study indicates that the blood salvaged intraoperatively contains elevated levels of complement split product and proinflammatory cytokines and that heparin-coated surfaces of the salvage device do not significantly influence the formation of inflammatory mediators.

Characterization of C4d Immunostaining Utilizing Paraffin-Embedded Tissue of Nonpresensitized Pediatric Heart Transplant Patients

Immunoperoxidase techniques for demonstration of complement split product C4d on paraffin-embedded endomyocardial biopsy samples have been used for the evaluation of humoral rejection mainly in adult heart transplantation. Interpretation of suspected humoral rejection in children requires knowledge of the pattern and frequency of C4d deposition in nonpresensitized pediatric heart transplantation patients. Paraffin-embedded endomyocardial biopsy samples of 65 patients with no rejection, acute cellular rejection (ACR), or early ischemic/reperfusion injury were studied. Six biopsies within each grade of ACR both early (<6 months) and late (>6 months) after heart transplantation were reviewed, as were 5 biopsies of ischemic/reperfusion injury. None of the subjects was sensitized prior to transplant. All slides were blindly evaluated for histologic features traditionally associated with humoral rejection. C4d staining was quantified by counting the number of positive capillaries in 10 random high-power fields (hpf). C4d positivity (C4d+) was defined as >10 capillaries/10 hpf. C4d+ was observed in 6 (9%) endomyocardial biopsy samples; 2 in the early and 4 in the late ACR groups. Four had ACR grades 1A/B, and 2 had grade 3B/4. Thirteen (20%) endomyocardial biopsy samples had histologic features suggestive of humoral rejection. Of these, only 2 were C4d+ and both had ACR grade 3B/4. No C4d+ staining was found in the ischemic/reperfusion injury group. C4d immunostaining was negative in endomyocardial biopsy samples of the majority of our patients (91%). Endomyocardial biopsy samples with C4d+ did not show consistent ACR grade or time specificity. Contrary to previously reported data, none of the ischemic/reperfusion injury endomyocardial biopsy samples was C4d+, and histologic features alone were poor predictors of C4d+.

The Role of Complement and VEGF on SDF‐1 Mediated Migration of CD34+ Stem Cells

CD34+ stem cells migrate from the bone marrow niche to sites of injury in response to various chemokines. We have investigated the influence of VEGF and complement peptides C3a and C5a, on the stromal derived factor‐1(SDF‐1)‐dependent migration of freshly isolated (from G‐CSF mobilized mononuclear cell preparations) and cord‐blood derived CD34+ stem cells. Freshly isolated CD34+ cells migrated in a dose‐dependent manner in response to SDF‐1, but were unable to migrate to the complement peptides C3a or C5a alone. However, both complement split products augmented the migration of CD34+ cells in response to low concentrations of SDF‐1. This augmented migration behavior was abrogated when CD34+ cells were incubated with anti‐C3a or anti‐C5a antibodies. Flow cytometry demonstrated that CD34+ stem cell express both the C3a and C5a receptors, and anti‐receptor antibodies also mitigated this synergy. Freshly isolated CD34+ cells responded initially to VEGF, but this response diminished after 1–2 days In contrast, with cultured cord blood‐derived CD34+ cells, we observed enhanced migration to SDF‐1 in the presence of VEGF compared to SDF‐1 alone. In conclusion, the migration of CD34+ stem cells depends on their source and ex vivo treatment, and may be enhanced to ischemic tissues (where SDF‐1 is produced) under the influence of other inflammatory and angiogenic signals.

Complement Split Products C3a and C4a Are Early Markers of Acute Lyme Disease in Tick Bite Patients in the United States

Background: Current laboratory markers do not readily detect acute Lyme disease. We assessed the utility of complement and its split products as markers of Lyme disease in patients shortly after a tick bite. Methods: Thirty-one consecutive acute Lyme disease patients, 14 with and 17 without erythema migrans (EM) skin rash, seen by a physician within 96 h of a tick bite were matched with 24 consecutive tick bite patients without Lyme disease symptoms and 46 healthy control subjects. Complement and split products measured included factor B, Bb, C4, C3c, C3a_{des Arg}, C4a_{des Arg}, C1q- and C3d-containing immune complexes, and C2. Results: C2, C4, C3 and factor B levels were within normal ranges in all groups. C3a and C4a levels were significantly higher in acute Lyme disease patients than in tick bite and healthy control groups (both p < 0.001). All acute Lyme disease patients, regardless of EM, had elevated levels of C3a or C4a. Few tick bite controls had elevated levels of C3a (2/20) or C4a (5/24) and only 1 of the healthy control subjects had elevated C3a (0/46) or C4a (1/32). Conclusions: These findings suggest that C3a and C4a may be useful markers of Lyme disease in patients seen shortly after tick bite, even in those without EM.

Emergency Department Treated Asthmatics have Elevated Levels of Plasma Complement Split Products (CSP) and Peripheral Blood CSP Receptor (CSPr) Levels Greater than those from Outpatient Asthmatics

Emergency Department Treated Asthmatics have Elevated Levels of Plasma Complement Split Products (CSP) and Peripheral Blood CSP Receptor (CSPr) Levels Greater than those from Outpatient Asthmatics

Treatment With Riboflavin and Ultraviolet Light Prevents Alloimmunization to Platelet Transfusions and Cardiac Transplants

Functional leukocytes in blood transfusions can cause alloimmunization. Previous studies have shown that exposure of platelet concentrates to riboflavin and light (Mirasol PRT treatment) causes irreparable modification of nucleic acids. This treatment does not interfere with platelet function but does inhibit a wide range of immunological functions of leukocytes present in platelet concentrates. The current study evaluated the ability of Mirasol treatment to prevent alloimmunization by platelet transfusions in rats. Lewis rats received eight transfusions of untreated or Mirasol-treated platelets containing leukocytes from DA rats. Animals were subsequently challenged with a heart transplant under cyclosporine treatment. Mirasol treatment caused apoptosis of the leukocytes as measured by annexin V and CD45 staining. Complement split products were deposited on the apoptotic bodies in the platelet pack. The primary and secondary immunoglobulin (Ig) M and IgG responses in rats that received Mirasol-treated platelets were almost completely abolished compared to animals that received untreated platelets. Untreated platelet transfusions elicited strong IgG responses that were associated with rapid rejection of subsequent heart transplants. Rejected hearts contained macrophage infiltrates and C4d deposits. In contrast, no grafts were rejected by recipients transfused with Mirasol-treated platelets. Macrophage infiltrates and C4d deposits were decreased in these grafts. Recipients that were presensitized to untreated platelets were capable of producing a memory response to Mirasol-treated platelets that caused accelerated rejection of subsequent transplants. Transfusions of platelets that were treated with riboflavin and ultraviolet light prevented presensitization to transplants. However, Mirasol-treated platelets were immunogenic in presensitized recipients.

Elevated levels of the complement split product Bb in early pregnancy are associated with subsequent development of pre-eclampsia

Elevated levels of the complement split product Bb in early pregnancy are associated with subsequent development of pre-eclampsia

A Comparative Analysis Between Survivors and Nonsurvivors with Antibody Mediated Cardiac Allograft Rejection

Antibody mediated rejection (AMR) is an important cause of graft loss in the post heart transplant period. The following study was conducted to determine differences between survivors and nonsurvivors who developed post heart transplant AMR. We retrospectively reviewed the charts of patients who received a heart transplant between January 1993 and December 2002. Patients with biopsy proven AMR were identified. This group was divided into survivors and nonsurvivors. Groups were compared with regards to demographics, T-cell flow panel of reactive antibodies (PRA), flow cross-matches (anti-donor HLA Class I and II), and short- and long-term outcomes. Results of endomyocardial biopsies were collected to allow calculation of sensitivity, specificity, negative- and positive predictive values as well as accuracy of immunoglobulins and complement split products in association to death. A total of 65 patients (8.9%) were diagnosed with AMR. Mean age was 48 y (range: 8-68 y) and 53.8% were males. Episodes of hemodynamic instability associated with AMR were observed in 37% of patients. Only two deaths were directly attributed to acute AMR. Nearly 20% of AMR patients developed transplant coronary artery disease. Univariate analysis identified T-PRA (P < 0.001), mean T-cell molecules of equivalent soluble fluorochrome (MESF) (P < 0.001) and mean B-cell MESF (P < 0.001) as possible factors associated with death. Neither demographics of complement split products were associated to late death. When studying patients with AMR, pretransplant T-PRA, T-cell, and B-cell MESF may identify individuals at risk of late death.

A novel inhibitor of the alternative pathway of complement reverses inflammation and bone destruction in experimental arthritis

Complement is an important component of the innate and adaptive immune response, yet complement split products generated through activation of each of the three complement pathways (classical, alternative, and lectin) can cause inflammation and tissue destruction. Previous studies have shown that complement activation through the alternative, but not classical, pathway is required to initiate antibody-induced arthritis in mice, but it is unclear if the alternative pathway (AP) plays a role in established disease. Previously, we have shown that human complement receptor of the immunoglobulin superfamily (CRIg) is a selective inhibitor of the AP of complement. Here, we present the crystal structure of murine CRIg and, using mutants, provide evidence that the structural requirements for inhibition of the AP are conserved in human and mouse. A soluble form of CRIg reversed inflammation and bone loss in two experimental models of arthritis by inhibiting the AP of complement in the joint. Our data indicate that the AP of complement is not only required for disease induction, but also disease progression. The extracellular domain of CRIg thus provides a novel tool to study the effects of inhibiting the AP of complement in established disease and constitutes a promising therapeutic with selectivity for a single complement pathway.

The Phosphatidylinositol 3-Kinase Signaling Pathway Exerts Protective Effects during Sepsis by Controlling C5a-Mediated Activation of Innate Immune Functions

The PI3K/Akt signaling pathway has been recently suggested to have controversial functions in models of acute and chronic inflammation. Our group and others have reported previously that the complement split product C5a alters neutrophil innate immunity and cell signaling during the onset of sepsis and is involved in PI3K activation. We report in this study that in vivo inhibition of the PI3K pathway resulted in increased mortality in septic mice accompanied by strongly elevated serum levels of TNF-alpha, IL-6, MCP-1, and IL-10 during sepsis as well as decreased oxidative burst activity in blood phagocytes. PI3K inhibition in vitro resulted in significant increases in TLR-4-mediated generation of various proinflammatory cytokines in neutrophils, whereas the opposite effect was observed in PBMC. Oxidative burst and phagocytosis activity was significantly attenuated in both neutrophils and monocytes when PI3K activation was blocked. In addition, PI3K inhibition resulted in strongly elevated TLR-4-mediated generation of IL-1beta and IL-8 in neutrophils when these cells were co-stimulated with C5a. C5a-induced priming effects on neutrophil and monocyte oxidative burst activity as well as C5a-induced phagocytosis in neutrophils were strongly reduced when PI3K activation was blocked. Our data suggest that the PI3K/Akt signaling pathway controls various C5a-mediated effects on neutrophil and monocyte innate immunity and exerts an overall protective effect during experimental sepsis.

Determinants of the Complement-Fixing Ability of Recipient Presensitization Against HLA Antigens

The presence of preformed alloantibodies with the ability to activate complement may pose a particular risk for kidney allograft rejection. The aim of this study was to evaluate variables that determine the complement-fixing capability of human leukocyte antigen (HLA) sensitization. Sixty-five sensitized patients with > or =10% pretransplant panel-reactive antibody (PRA) levels uncovered by immunoglobulin G [IgG]FlowPRA HLA class I and/or class II screening were included. Applying modified FlowPRA screening, sera were evaluated for patterns of alloreactive IgG subclasses and IgM, and, in parallel, for their complement-activating ability assessed by flow cytometric detection of human complement split product deposition ([C4d]FlowPRA). Approximately two-thirds (68%) of tested sera were found to contain complement-fixing alloreactivity (> or =10%[C4d]FlowPRA). IgG1 type panel reactivity was predominant (detectable HLA class I and II reactivity in 93% and 91% of IgG-positive sera), followed by IgG3 (49%/44%), IgG2 (44%/27%), and IgG4 (19%/11%). Applying partial correlation we found an independent correlation of both %[IgG1]FlowPRA and %[IgG3]FlowPRA with %[C4d]FlowPRA reactivities (P< or =0.01). In addition, for IgG1 a contribution of the amount of bound alloantibody to complement-fixation was observed. Complement-fixation was also favored by the simultaneous presence of alloreactive IgG1, IgG3, and IgM. Previous grafting, but not pregnancy and transfusion, was independently associated with complement-fixing sensitization (P<0.05), presumably due to increased IgG1 type reactivity. Anti-HLA antibody-triggered complement activation is dependent on both the pattern of Ig reactivities and the amount of bound antibody. Previous transplantation represents a major risk factor for the development of complement-fixing sensitization.

Antibody and Complement in Transplant Vasculopathy

Advances in immunosuppression have decreased the incidence of acute rejection, but the development of vasculopathy in the coronary arteries of transplants continues to limit the survival of cardiac allografts. Transplant vasculopathy has also been referred to as accelerated graft arteriosclerosis because it has features of arteriosclerosis, but it is limited to the graft and develops over a period of months to years. Although the pathological features of transplant vasculopathy are well defined, the causative mechanisms are not completely understood. This review focuses on the mechanisms by which antibody and complement can cause or contribute to coronary vasculopathy in cardiac transplants. Antibodies and complement can have independent effects, but the combination of antibodies and complement with inflammatory cells has greater pathogenic potential for the endothelial and smooth muscle cells of the coronary arteries. For example, stimulation through receptors for IgG or complement split products can activate macrophages, but stimulation through combinations of these receptors generates synergistic results. Together, antibodies and complement efficiently integrate the activation of endothelial cells, platelets, and macrophages, which are 3 of the primary components in the pathogenesis of transplant vasculopathy. Recent findings indicate that antibodies and complement produced within the transplant may contribute to vascular pathology in some transplants. Acute rejection caused by antibodies and complement has been treated by combinations of plasmapheresis, intravenous gamma-globulin and monoclonal antibodies to CD20 on B lymphocytes. The effect of these treatment modalities on the development of coronary vasculopathy is unknown.

196: Pattern of C4d staining is important in cardiac transplantation

Purpose: Staining for the complement split product, C4d has been used to detect possible antibody mediated rejection (AMR) in cardiac transplantation. Staining with immunohistochemical (IHC) techniques has been associated with clinical AMR. However, until now there have been only qualitative descriptions of C4d staining. We analyzed C4d staining to describe the pattern of staining with correlation to cardiac filling pressures.

Antibody to human leukocyte antigen triggers endothelial exocytosis.

Although antibodies to HLA play a role in the pathogenesis of diseases processes such as rejection of transplanted organs, the precise mechanisms by which antibodies cause tissue injury are not completely understood. We hypothesized that antibodies to host tissues cause inflammation in part by activating endothelial exocytosis of granules that contain prothrombotic mediators such as von Willebrand Factor (VWF) and proinflammatory mediators such as P-selectin. To test this hypothesis, we treated human endothelial cells with murine monoclonal antibody W6/32 to HLA class I and then measured exocytosis by the release of VWF and the externalization of P-selectin. Antibody to HLA activates endothelial exocytosis in a dose-dependent manner over time. The biologically active complement split product, C5a, adds a slight but significant increase to antibody induction of exocytosis. Antibody to HLA alone or with C5a did not damage the cells. Cross-linking of HLA appears to play a role in the ability of antibody to activate exocytosis, because the W6/32 monovalent Fab fragment did not activate VWF release, but the bivalent Fab'2 was effective in triggering exocytosis. To explore the in vivo effects of antibody upon graft injury, we infused W6/32 Fab'2 antibody to human HLA into severe combined immunodeficient/beige mice that had been transplanted with human skin grafts. Antibody to HLA activated exocytosis and inflammation in human skin grafts. Our data show that antibody to host antigens can activate human endothelial cell exocytosis and leukocyte trafficking. By triggering vascular inflammation, antibody activation of exocytosis may play a role in transplant rejection.

Relation of Elevated Plasma Complement Split Product Levels to Their Peripheral Blood Leukocyte Receptors and Asthma Severity

RATIONALE: C5a levels are proposed to have a protective role in induction of allergic inflammation while contributing to established allergic processes (Kohl). This study determines the relation of peripheral blood complement split product receptors (CSPr) for C5a desArg and C3a desArg with plasma levels of CSP and adult asthma severity.

Effect of Emergency Room Treatment of Acute Asthma on Plasma Levels of Complement Split Products

Effect of Emergency Room Treatment of Acute Asthma on Plasma Levels of Complement Split Products

Unravelling in vitro variables of major importance for the outcome of mass spectrometry-based serum proteomics

The use of mass spectrometry (MS) for analysing low-molecular weight proteins and peptides from biological fluids has a great, yet not fully realized, potential for biomarker discovery. To prune MS-data as much as possible for non-relevant non-biological variation the development of standardized protocols for handling and processing the samples before MS and adjusting data after MS to compensate for method-induced variability are warranted. This calls for knowledge about how different variables contribute to MS-based proteome analyses. In addition, identification of the peptides involved in pre-analytical variation will be helpful in evaluating the clinical significance of predictive models derived from MS data. Using human sera, extraction by weak cation-exchange magnetic beads, and analysis by MALDI-TOF MS we here evaluated pre-analytical variation and identify peptides involved in this. The influences of humidity, temperature, and time for preparation of sera on spectral changes were evaluated. Also, the reproducibility of the methods and the effect of a baseline correction procedure were examined. Low temperatures, short handling times, and a baseline correction procedure minimize the contribution of artifacts to sample variability as observed by MS. The complement split product C3f and fragments thereof appear to be sensitive indicators of sample handling induced modifications. Other peptides that are indicative of such variability are fibrin and kininogen fragments. Using strict experimental guidelines as well as standardized sample collection procedures it is possible to obtain reproducible peak intensities and positions in serum mass profiling using magnetic bead-based fractionation and MALDI-TOF MS.

Complement split product C4d isolated detection on human erythrocytes in antibody-mediated allo-transplant rejection: A new bystander effect and a potential non-invasive blood test for humoral rejection

Complement split product C4d isolated detection on human erythrocytes in antibody-mediated allo-transplant rejection: A new bystander effect and a potential non-invasive blood test for humoral rejection

Pivotal Role of Complement-Fixing HLA Alloantibodies in Presensitized Kidney Allograft Recipients

Recipient presensitization represents a major risk factor for kidney allograft loss. Complement fixation may be a critical attribute of deleterious alloantibodies. We investigated clinical impact of complement-fixing HLA presensitization employing [C4d]FlowPRA, a novel assay permitting selective detection of HLA panel reactive antibody (PRA)-triggered C4 complement split product deposition. A cohort of 338 kidney transplants was evaluated for presensitization applying [C4d]FlowPRA together with [IgG]FlowPRA and complement-dependent cytotoxicity (CDC)-PRA. Analysis of HLA class I alloreactivities revealed a high incidence of C4d-positive graft dysfunction in [IgG]FlowPRA(+)/[C4d]FlowPRA(+) and [IgG]FlowPRA(+)/[C4d]FlowPRA(-) recipients (23% and 22% vs. 3% in [IgG]FlowPRA(-) patients). Only patients with complement-fixing HLA class I immunization had inferior graft survival [75% (3 years) vs. 91% and 89%, respectively (p=0.036)]. Despite frequent finding of capillary C4d deposition (28%), complement-fixing HLA class II immunization was not associated with inferior survival rates. This may have been due to reduction of clinical effects by intense immunosuppression in presensitized patients. Evaluating CDC, 29% of CDC-PRA(+)/[C4d]FlowPRA(+) recipients had C4d-positive graft dysfunction. For these patients 3-year graft survival was worst, followed by CDC-PRA(+)/[C4d]FlowPRA(-) and CDC-PRA(-) patients (76% vs. 81% vs. 90%, p=0.014). Results highlight a strong impact of complement-fixing HLA presensitization. Discerning complement-activating abilities of HLA alloantibodies, [C4d]FlowPRA may help identify recipients at particularly high risk for graft rejection and loss.