Plasma levels of soluble membrane attack complex are elevated despite viral suppression in HIV patients with poor immune reconstitution.

The complement system is now a therapeutic target for the management of serious and life-threatening conditions such as paroxysmal nocturnal hemoglobinuria, atypical hemolytic uremic syndrome, glomerulonephritis and other diseases caused by complement deficiencies or genetic variants. As complement therapeutics expand into more clinical conditions, monitoring complement activation is increasingly important, as is the baseline levels of complement activation fragments in blood or other body fluid levels. Although baseline complement levels have been reported in the literature, the majority of these data were generated using non-standard assays and with variable sample handling, potentially skewing results. In this study, we examined the plasma and serum levels of the soluble membrane attack complex of complement (sMAC). sMAC is formed in the fluid phase when complement is activated through the terminal pathway. It binds the regulatory proteins vitronectin and/or clusterin and cannot insert into cell membranes, and can serve as a soluble diagnostic marker in infectious disease settings, as previously shown for intraventricular shunt infections. Here we show that in healthy adults, serum sMAC levels were significantly higher than those in plasma, that plasma sMAC levels were similar between in African Americans and Caucasians and that plasma sMAC levels increase with age. Plasma sMAC levels were significantly higher in virally suppressed people living with HIV (PLWH) compared to non-HIV infected healthy donors. More specifically, PLWH with CD4+ T cell counts below 200 had even greater sMAC levels, suggesting diagnostic value in monitoring sMAC levels in this group.

Membrane attack complex generation increases as a function of time in stored blood.

OBJECTIVE: To determine if the complement system, a potent mediator of inflammation, contributes to haemolysis during red blood cell (RBC) storage. BACKGROUND: RBCs in storage undergo structural and biochemical changes that may result in adverse patient outcomes post-transfusion. Complement activation on leukodepletion and during storage may contribute to the RBC storage lesion. METHODS/MATERIALS: We performed a cross-sectional analysis of aliquots of leukoreduced RBC units, stored for 1-6 weeks, for the levels of C3a, C5a, Bb, iC3b, C4d and C5b-9 [membrane attack complex (MAC)] by enzyme-linked immunosorbent assay (ELISA). RESULTS: We observed that only MAC levels significantly increased in RBC units as a function of storage time. We also observed that the level of C5b-9 bound to RBCs increased as a function of storage time. CONCLUSION: MAC levels increased over time, suggesting that MAC is the primary complement-mediated contributor to changes in stored RBCs. Inhibition of the terminal complement pathway may stabilise RBC functionality and extend shelf life.

Deletion of the complement phagocytic receptors CR3 and CR4 does not alter susceptibility to experimental cerebral malaria.

Complement receptors for C3-derived fragments (CR1-4) play critical roles in innate and adaptive immune responses. Of these receptors, CR3 and CR4 are important in binding and phagocytosis of complement-opsonized pathogens including parasites. The role of CR3 and CR4 in malaria or in cerebral malaria (CM) has received little attention and remains poorly understood in both human disease and rodent models of malaria. CR3 and CR4 are members of the ß(2) -integrin family of adhesion molecules and are expressed on all leucocytes that participate in the development of CM, most importantly as it relates to parasite phagocytosis (monocytes/macrophages) and antigen processing and presentation (dendritic cells). Thus, it is possible that these receptors might play an important role in disease development. To address this question, we examined the role of CR3(-/-) and CR4(-/-) in experimental cerebral malaria (ECM). We found that both CR3(-/-) and CR4(-/-) mice were fully susceptible to ECM and developed disease comparable to wild-type mice. Our results indicate that CR3 and CR4 are not critical to the pathogenesis of ECM despite their role in elimination of complement-opsonized pathogens. These findings support recent studies indicating the importance of the terminal complement pathway and the membrane attack complex in ECM pathogenesis.

Deletion of carboxypeptidase N delays onset of experimental cerebral malaria.

Complement contributes to inflammation during pathogen infections; however, less is known regarding its role during malaria and in the severest form of the disease, cerebral malaria. Recent studies have shown that deletion of the complement anaphylatoxins receptors, C3aR and C5aR, does not alter disease susceptibility in experimental cerebral malaria (ECM). This does not, however, preclude C3a- and C5a-mediated contributions to inflammation in ECM and raises the possibility that carboxypeptidase regulation of anaphylatoxin activity rapidly over rides their functions. To address this question, we performed ECM using carboxypeptidase N-deficient (CPN(-/-)) mice. Unexpectedly, we found that CPN(-/-) mice survived longer than wild-type mice, but they were fully susceptible to ECM. CD4(+) and CD8(+) T cell infiltration was not reduced at the peak of disease in CPN(-/-) mice, and there was no corresponding reduction in pro-inflammatory cytokine production. Our results indicate that carboxypeptidases contribute to the pathogenesis of ECM and that studies examining the contribution of other carboxypeptidase families and family members may provide greater insight into the role these enzymes play in malaria.

Complement anaphylatoxin receptors on neurons: new tricks for old receptors?

Activation of the complement system has been reported in a variety of inflammatory diseases and neurodegenerative processes of the CNS. Recent evidence indicates that complement proteins and receptors are synthesized on or by glial cells and, surprisingly, neurons. Among these proteins are the receptors for the chemotactic and anaphylactic peptides, C5a and C3a, which are the most-potent mediators of complement inflammatory functions. The functions of glial-cell C3a and C5a receptors (C3aR and C5aR) appear to be similar to immune-cell C3aRs and C5aRs. However, little is known about the roles these receptors might have on neurons. Indeed, when compared with glial cells, neurons display a distinct pattern of C3aR and C5aR expression, in either the normal or the inflamed CNS. These findings suggest unique functions for these receptors on neurons.

Transforming growth factor-beta2-mediated regulation of C3 gene expression in monocytes.

In this report, we show that transforming growth factor-beta2 (TGF-beta2) regulates C3 gene expression in the human monocyte cell lines, U937 and THP-1, and human peripheral blood monocytes. Treatment of U937 or THP-1 cells with TGF-beta2 resulted in a dose-dependent induction of C3 protein and mRNA expression. Dose-dependent increases of C3 protein and mRNA levels were also detected in TGF-beta2-treated primary blood monocytes, demonstrating that TGF-beta2 can modulate C3 expression in nontransformed monocytes. Kinetic analysis demonstrated that TGF-beta2-mediated induction of C3 mRNA and protein could be detected within 8 hr, and the induction was continuous up to 72 hr. Exposure of cells to TGF-beta2 for as little as 2 hr was sufficient to induce C3 expression. TGF-beta2 did not significantly increase C3 mRNA stability as determined by mRNA half-life studies. Collectively, our results demonstrate that TGF-beta2 regulates the expression of C3 in monocytes and suggest that TGF-beta2 may play a role in modulating the synthesis of C3 during inflammatory responses.

Transforming growth factor-beta2 regulates C3 secretion in monocytes through a protein kinase C-dependent pathway.

Previously, we reported that TGF-beta2 regulates the C3 gene expression in a dose- and time-dependent manner in monocytes. To extend these studies, we examined the role of PKC in the TGF-beta2-mediated induction of C3 expression by the human monocyte cell line, U937. Treatment of U937 cells with the PKC inhibitors, H7 and calphostin C, suppressed TGF-beta2-mediated induction of C3 protein levels, but not mRNA levels, in a dose-dependent manner. At the highest concentrations of H7 and calphostin C, C3 protein levels were inhibited 50% and 93%, respectively, compared to control levels. Treatment of U937 cells with HA1004, a weak PKC inhibitor used as a control for H7, did not inhibit induction of C3 protein levels. Down-modulating PKC with a prolonged exposure of U937 cells to PMA also suppressed TGF-beta2-mediated C3 protein induction by as much as 82%. Incubating cell extracts isolated from TGF-beta2-treated U937 cells with the PKC substrate, MIBP(4-14), resulted in increased substrate phosphorylation compared to cell extracts isolated from untreated cells. Addition of calphostin C suppressed the increased substrate phosphorylation by TGF-beta2. Furthermore, biosynthetic labeling of U937 cells treated with TGF-beta2 and calphostin C demonstrated an accumulation of C3 protein within cell lysates compared to controls. Collectively, these studies suggest a role for PKC in the secretion of C3 protein during TGF-beta2-mediated regulation of C3 expression in U937 cells.

Quantitation of complement factor D in human serum by a solid-phase radioimmunoassay.

A sensitive solid-phase radioimmunoassay is described which quantitates human D to 1-2 ng/ml. The assay was used to measure the concentration of D in normal and acute-phase sera and sera from individuals with systemic lupus erythematosus. All 3 groups of sera had comparable levels of D with mean values of 1.8, 2.3 and 2.5 micrograms/ml, respectively. Also tested were sera decomplemented in vitro by activators of the classical and alternative pathways. The results indicated that D is not depleted by alternative or classical pathway activation. However, heat inactivation (56 degrees C, 30 min) of serum resulted in almost complete loss of antigenic D.

Expression of decay-accelerating factor (CD55), membrane cofactor protein (CD46) and CD59 in the human astroglioma cell line, D54-MG, and primary rat astrocytes.

In this report, we have shown the expression of the complement regulatory proteins decay-accelerating factor (DAF, CD55), membrane cofactor protein (MCP, CD46) and CD59 on human D54-MG astroglioma cells by several methods, including immunofluorescence, flow cytometry and Western blotting and Northern blot analysis. These studies demonstrate that all three proteins are structurally and antigenically similar to their counterparts expressed on HepG2 and SW480 cells (hepatocyte and epithelial cell lines, respectively). D54-MG cells express mRNA for all three proteins of the appropriate size(s). The phosphatidylinositol-specific enzyme, PIPLC, cleaved DAF from the surface of D54-MG cells, demonstrating that DAF is linked by a glycophospholipid anchor as has been shown for other cell types. Flow cytometry demonstrates that primary rat astrocytes also constitutively express all three regulatory proteins. These data are the first to demonstrate the expression of CD59 on astrocytes, and the presence of all three regulatory proteins on astrocytes suggests that regulation of complement activation in the central nervous system is important in neural host defense mechanisms.

Kinetics of anaphylatoxin C5a receptor expression during experimental allergic encephalomyelitis.

In this study, we investigated the expression of the C5aR in spinal cords of Lewis rats with experimental allergic encephalomyelitis (EAE). Using in situ hybridization (ISH) we analyzed the kinetics of C5aR at different time points of EAE (preclinical stage, clinical peak, remission phase). We observed that C5aR mRNA was readily detected in the CNS of EAE rats at all the stages of the disease. Using a combination of ISH and immunohistochemistry, we formally demonstrated that C5aR is strongly expressed on microglial cells and hypertrophic astrocytes during EAE. The potential involvement of C5a receptor in EAE physiopathology is discussed.

Interferon-gamma regulation of C3 gene expression in human astroglioma cells.

In this report, we show that the human astroglioma cell line, D54-MG, constitutively expresses C3 mRNA and secretes antigenically detectable C3 protein. The cytokine interferon-gamma (IFN-gamma) enhances C3 mRNA and protein expression by D54-MG cells in a dose- and time-dependent manner. C3 mRNA from both D54-MG cells and primary human adult astrocytes has the same apparent size (5.1-5.2 kb) as C3 mRNA from hepatocyte and monocyte cell lines. Constitutive C3 mRNA levels in D54-MG cells and primary human astrocytes are comparable. Primary rat astrocytes also constitutively express C3 mRNA, which is enhanced upon exposure to IFN-gamma. These data are novel since expression of C3 in other cell types is refractory to IFN-gamma. In the central nervous system (CNS), endogenous complement production by astrocytes, and enhancement by the cytokine IFN-gamma, may contribute to the pathogenesis of inflammatory demyelinating diseases such as multiple sclerosis (MS).

Expression of the receptors for the C5a anaphylatoxin, interleukin-8 and FMLP by human astrocytes and microglia.

The expression of chemotactic receptors in the central nervous system is largely unexplored. In this study, we examined human astrocytes and microglia as well as the conditionally immortalized human astrocyte cell line HSC2 for expression of the C5a-anaphylatoxin receptor (C5aR), the interleukin-8 receptor (IL-8R) and the f-Met-Leu-Phe receptor (FMLPR). Using flow cytometry, indirect immunofluorescence and RT-PCR analysis, we demonstrated that astrocytes, microglia and HSC2 cells contain specific RNA and express surface protein for all three chemotactic receptors. These are the first studies to demonstrate definitively the expression of these chemotactic receptors astrocytes and microglia, thereby expanding the types of cells known to express chemotactic receptors. Moreover, these data suggest that these chemotactic receptors may play an important role in mediating the inflammatory response and perhaps other yet undescribed biological phenomena in the central nervous system.

Elevated levels of the complement components C3 and factor B in ventricular cerebrospinal fluid of patients with traumatic brain injury.

Immunological events occurring in the central nervous system (CNS) as a result of head trauma are largely unexplored. We report here that the levels of the alternative pathway complement proteins C3 and factor B are elevated in the cerebrospinal fluid (CSF) of head-injured patients. C3 and factor B indices suggest that changes in C3 and factor B levels in CSF are most likely due to altered blood-brain barrier integrity and not to intrathecal synthesis. These data demonstrate, for the first time, elevated levels of complement proteins in CSF of patients with severe traumatic brain injury. Elevated complement levels in brain injury may contribute to secondary damage.

Intracerebral complement C5a receptor (CD88) expression is regulated by TNF and lymphotoxin-alpha following closed head injury in mice.

The anaphylatoxin C5a is a potent mediator of inflammation in the CNS. We analyzed the intracerebral expression of the C5a receptor (C5aR) in a model of closed head injury (CHI) in mice. Up-regulation of C5aR mRNA and protein expression was observed mainly on neurons in sham-operated and head-injured wild-type mice at 24 h. In contrast, in TNF/lymphotoxin-alpha knockout mice, the intracerebral C5aR expression remained at low constitutive levels after sham operation, whereas it strongly increased in response to trauma between 24 and 72 h. Interestingly, by 7 days after CHI, the intrathecal C5aR expression was clearly attenuated in the knockout animals. These data show that the posttraumatic neuronal expression of the C5aR is, at least in part, regulated by TNF and lymphotoxin-alpha at 7 days after trauma.

Experimental diffuse axonal injury induces enhanced neuronal C5a receptor mRNA expression in rats.

Several studies suggest the involvement of the complement system in the pathophysiology of traumatic brain injury (TBI). Since the intrathecal generation of anaphylatoxin C5a has been shown to mediate inflammatory effects within the central nervous system, we sought to characterize the cellular expression of the mRNA for the C5a receptor (C5aR, CD88) in brains of rats with experimental diffuse axonal injury (DAI) by in situ hybridization. Infiltrating leukocytes expressing C5aR mRNA were seen in meninges and lateral ventricles as early as 4 h after induction of DAI. The number of infiltrating C5aR-positive cells increased gradually up to 24 h after trauma. Within the brain parenchyma, up-regulation of C5aR mRNA expression was first seen in cerebellar Purkinje cells within 8 h. At 24 h after TBI, expression of C5aR mRNA was widespread bilaterally throughout the cortex and cerebellum, the cellular expression being restricted to pyramidal neurons and Purkinje cells. The intensity of C5aR transcript signals on neurons increased further up to 96 h after trauma. Ligand binding of C5a to its receptor on neurons might mediate previously unknown functions, thus possibly leading to neurotoxicity and secondary neuronal damage after TBI.

Expression of the anaphylatoxin C5a receptor in the oligodendrocyte lineage.

Expression of the C5a receptor in the central nervous system has been demonstrated on microglia, astrocytes and neurons. In the present study, we demonstrate C5aR expression in vitro by rat and murine O2-A progenitor cells and oligodendrocytes. We also observed that in vitro differentiation of O2-A progenitors into mature oligodendrocytes is accompanied by down-regulation of C5aR mRNA expression. These results suggest that the C5aR may be a marker for oligodendroglial differentiation and play a role in oligodendrocyte function.

Differential regulation of C3 gene expression in human astroglioma cells by interferon-gamma and interleukin-1 beta.

In this report, we examined interferon-gamma (IFN-gamma) and interleukin-1 beta (IL-1 beta)-mediated regulation of the expression of C3, the third component of complement, in a human astroglioma cell line. Interleukin-1 beta induced C3 protein expression ten-fold more rapidly than IFN-gamma. De novo protein synthesis was required for IFN-gamma to stimulate C3 expression, while cycloheximide and IL-1 beta treatment of cells markedly increased C3 expression. Actinomycin D, inhibited C3 gene induction by IFN-gamma and IL-1 beta suggesting that these cytokines act, in part, at the transcriptional level to enhance C3 expression. Understanding cytokine-mediated regulation of complement gene expression in the astrocyte is important in defining the role of these molecules in CNS inflammation and autoimmune diseases.

Enhanced expression of chemotactic receptors in multiple sclerosis lesions.

We have previously shown that astrocytes and microglia express the receptors for C5a, interleukin-8 (IL-8) and N-formyl-Met-Leu-Phe (FMLP) in vitro. The expression and function of chemotactic receptors in the central nervous system (CNS) is, however, largely unexplored. In this study, we examined tissue sections from normal human brain and active, chronic active and chronic silent multiple sclerosis (MS) lesions for the expression of the receptors for C5a, IL-8 and FMLP by immunohistochemistry. In normal brain tissue, the expression of all three receptors was seen at low levels on astrocytes and microglia. In contrast, expression for all three receptors was markedly elevated on foamy macrophages in the acute and chronic active MS lesions. In addition, fibrous astrocytes stained intensely for the C5a receptor in the chronic active disease. Receptor expression in the chronic silent lesion was low and similar to that seen in normal brain, with staining confined to a few hypertrophic astrocytes and foamy macrophages. These are the first studies to demonstrate expression of these receptors in the CNS and elevated receptor expression in inflammatory MS lesions. The data suggest that these chemotactic receptors may play a role in inflammatory responses in MS and possibly in other CNS diseases.