Cell Surface Complement Regulatory Proteins Research Articles

Exploitation of the complement system by oncogenic Kaposi's sarcoma-associated herpesvirus for cell survival and persistent infection.

During evolution, herpesviruses have developed numerous, and often very ingenious, strategies to counteract efficient host immunity. Specifically, Kaposi's sarcoma-associated herpesvirus (KSHV) eludes host immunity by undergoing a dormant stage, called latency wherein it expresses a minimal number of viral proteins to evade host immune activation. Here, we show that during latency, KSHV hijacks the complement pathway to promote cell survival. We detected strong deposition of complement membrane attack complex C5b-9 and the complement component C3 activated product C3b on Kaposi's sarcoma spindle tumor cells, and on human endothelial cells latently infected by KSHV, TIME-KSHV and TIVE-LTC, but not on their respective uninfected control cells, TIME and TIVE. We further showed that complement activation in latently KSHV-infected cells was mediated by the alternative complement pathway through down-regulation of cell surface complement regulatory proteins CD55 and CD59. Interestingly, complement activation caused minimal cell death but promoted the survival of latently KSHV-infected cells grown in medium depleted of growth factors. We found that complement activation increased STAT3 tyrosine phosphorylation (Y705) of KSHV-infected cells, which was required for the enhanced cell survival. Furthermore, overexpression of either CD55 or CD59 in latently KSHV-infected cells was sufficient to inhibit complement activation, prevent STAT3 Y705 phosphorylation and abolish the enhanced survival of cells cultured in growth factor-depleted condition. Together, these results demonstrate a novel mechanism by which an oncogenic virus subverts and exploits the host innate immune system to promote viral persistent infection.

1,25-Dihydroxyvitamin D3 Promotes a Sustained LPS-Induced NF-κB-Dependent Expression of CD55 in Human Monocytic THP-1 Cells

The vitamin D3 system imposes immunosuppressive effects on monocytic cells, in part, by inhibiting NF-κB-dependent expression of proinflammatory mediators. CD55, a cell surface complement regulatory protein that promotes protective and anti-inflammatory properties, is reportedly an NF-κB target gene transiently induced in monocytic cells by the bacterial endotoxin LPS. CD55 is elevated on white cells in women experiencing preterm labor (a pathophysiology commonly associated with bacterial infection) and failure to maintain CD55 was associated with subsequent preterm delivery. We examined the influence of vitamin D3 signaling on LPS-induced expression of CD55 in human monocytic THP-1 cells using quantitative PCR, immunoblot, immunohistochemistry, and NF-κB activation pathway inhibitors. Non-NF-κB targets CD14 and CD11b, which modulate bacterial surveillance and eradication, respectively, were also examined. LPS produced a rapid transient 1.6-fold increase in CD55 mRNA. 1,25-D3 alone did not affect CD55 mRNA expression within the first 48 h. However, in 1,25-D3 pretreated cells, LPS produced a >4-fold immediate and sustained increase in CD55 mRNA and protein expression, which was blocked by NF-κB inhibitors. Our results unexpectedly suggest that vitamin D3 signaling may promote an anti-inflammatory response through an NF-κB-dependent increase in CD55 expression. As expected, LPS or 1,25-D3 alone led to sustained increases in CD14 and CD11b expression. In 1,25-D3 pretreated cells, LPS differentially regulated protein expression - CD14 (21-fold increase) and CD11b (a transient 2-fold decrease) - principally at the posttranscriptional level. The coordinated temporal expression of CD55, CD14 and CD11b would contribute to an anti-inflammatory response by providing protection against complement-mediated cell lysis during pathogen recognition and eradication. Overall, the vitamin D3 system may play a role coordinating an anti-inflammatory response pattern of the host complement immune system. This may be particularly important when considering the high rates of preterm births in blacks, a population that exhibits reduced circulating vitamin D3 levels.

Absence of spermatozoal CD46 protein expression and associated rapid acrosome reaction rate in striped field mice (Apodemus agrarius)

BackgroundIn rodents, the cell surface complement regulatory protein CD46 is expressed solely on the spermatozoal acrosome membrane. Ablation of the CD46 gene is associated with a faster acrosome reaction. Sperm from Apodemus flavicollis (yellow-necked field mice), A. microps (pygmy field mice) and A. sylvaticus (European wood mice) fail to express CD46 protein and exhibit a more rapid acrosome reaction rate than Mus (house mice) or BALB/c mice. A. agrarius (striped field mice) belong to a different Apodemus subgenus and have pronounced promiscuity and large relative testis size. The aim of this study was to determine whether A. agrarius sperm fail to express CD46 protein and, if so, whether A. agrarius have a faster acrosome reaction than Mus.MethodsReverse transcription polymerase chain reaction (RT-PCR) was used to assess whether A. agrarius transcribe testicular CD46 mRNA. RT-PCR was supplemented with 3'- and 5'-rapid amplification of cDNA ends to determine the complete nucleotide sequence of A. agrarius CD46. Fluorescence microscopy was used to assess whether CD46 protein is expressed by A. agrarius sperm. The acrosome status of A. agrarius sperm was calculated over time by immunocytochemistry using peanut agglutinin lectin.ResultsWe demonstrate that A. agrarius mice transcribe two unique alternatively spliced testicular CD46 mRNA transcripts, both lacking exon 7, which differ from those described previously in other Apodemus species. The larger A. agrarius CD46 transcript has an insert between exons 10 and 11 which, if translated, would result in a novel cytoplasmic tail. In addition, A. agrarius CD46 transcripts have an extended AU-rich 3'-untranslated region (UTR) and a truncated 5'-UTR, resulting in failure to express spermatozoal CD46 protein. We show that A. agrarius has a significantly faster spontaneous acrosome reaction rate than A. sylvaticus and Mus.ConclusionAbsence of CD46 protein expression is associated with acrosomal instability in rodents. A. agrarius mice express novel CD46 transcripts, resulting in the trade of spermatozoal CD46 protein expression for a rapid acrosome reaction rate, in common with other species of field mice. This provides a strategy to increase competitive sperm advantage for individuals, leading to faster fertilisation in this highly promiscuous genus.

Rapid sperm acrosome reaction in the absence of acrosomal CD46 expression in promiscuous field mice (Apodemus)

There is pronounced promiscuity and sperm competition in long-tailed field mice (Apodemus sylvaticus). These mice have evolved unusual sperm behaviour favouring rapid fertilisation, including dynamic formation of sperm trains and their subsequent dissociation. The cell surface complement regulatory (CReg) protein CD46 is broadly expressed in eutherian mammals other than rodents, in which it is expressed solely on the spermatozoal acrosomal membrane. Ablation of the CD46 gene has been associated with a faster acrosome reaction (AR) rate in inbred laboratory mice. Here, we demonstrate that wild-caught field mice of three species, A. sylvaticus, A. flavicollis and A. microps, exhibit a more rapid AR than wild-caught house mice Mus musculus or inbred laboratory BALB/c mice. We also demonstrate that wild-caught field mice of these three species, unlike house mice, produce alternatively spliced transcripts of testicular CD46 mRNA lacking exons 5-7 or 6-7, together with an extended 3' - and often truncated 5'-utr, leading to failure to express any sperm CD46 protein in both the testis and epididymis. Male field mice may therefore have traded expression of this CReg protein for acrosomal instability, providing a novel genus-specific strategy to favour rapid fertilisation and competitive advantage in the promiscuous reproductive behaviour of wild field mice.

Expression of complement regulatory proteins on human natural killer cell subsets

The cell surface complement regulatory (CReg) proteins CD46, CD55 and CD59 are widely distributed on human leucocytes and protect against complement-mediated damage. To investigate heterogeneity in CReg protein expression by human natural killer (NK) cells, levels were assessed on resting and activated NK cell subsets identified phenotypically on the basis of expression of CD56 and CD158 markers. Levels of all three CReg proteins on CD56 + cells were lower than on T cells ( p < 0.05). Freshly isolated CD56 bright cells expressed higher levels of CD55 than CD56 dim cells ( p < 0.05). CD158a + cells expressed significantly lower levels of both CD46 and CD59, and CD158e + cells expressed significantly lower levels of CD46, than CD158a − CD158e − cells, respectively (both p < 0.05). Stimulation with PHA did not significantly alter NK cell surface CReg protein levels whereas, following culture with IL-2, CD46 and CD59 were decreased on both CD56 bright and CD56 dim subsets ( p < 0.05). In the case of CD59, this was independent of T cells. Only CD46 was significantly downregulated on CD158b + (GL183 +) and CD158e (NKB1 +) subsets ( p < 0.05). However, culture in IL-15 significantly increased levels of all three CReg proteins. These observations that CReg proteins are downregulated on certain NK cell subsets following activation with IL-2 are opposite to previous findings for other leucocyte subpopulations. Activated NK cells may instead use other strategies for protection against complement-mediated damage in a local inflammatory response.

Levels of expression of complement regulatory proteins CD46, CD55 and CD59 on resting and activated human peripheral blood leucocytes

The cell surface complement regulatory (CReg) proteins CD46, CD55 and CD59 are widely expressed on human lymphoid and non-lymphoid cells. This study aimed to compare systematically levels of CReg expression by different leucocyte subsets and to determine whether levels were increased following activation in vitro. Levels of each CReg protein were similar on freshly isolated monocytes and all major lymphocyte subsets, except that CD4(+) cells expressed significantly less CD46 than CD8(+) cells (P < 0.05) while the reverse was observed for CD55 (P < 0.02). CD56(+) cells, predominantly natural killer cells, expressed significantly lower levels of CD59 than T cells (P < 0.02). CD45RO(+) cells had higher levels of surface CD46 and CD59, but lower levels of CD55, than CD45RO(-) cells (P < 0.02); CD25(+) cells also expressed significantly less CD55 than CD25(-) cells (P < 0.002). Neutrophils expressed higher levels of CD59, but lower levels of CD55, than monocytes. Following activation with phytohaemagglutinin, CD46 was up-regulated on all leucocyte subsets with the exception of CD56(+) cells. Both CD55 and CD59 were also markedly up-regulated on monocytes, and CD55 expression was greater on CD8(+) than CD4(+) cells following activation (P < 0.02). Lipopolysaccharide treatment did not significantly alter B-cell expression of CReg proteins whereas CD55 and CD59, but not CD46, were significantly up-regulated on monocytes (P < 0.02). These observations that CReg proteins are up-regulated on certain activated leucocyte subsets indicate that levels would be increased following immune responses in vivo. This could enhance both protection against local complement activation at inflammatory sites and also the immunoregulatory properties of these leucocytes.

Elevated serum levels of soluble membrane cofactor protein (CD46, MCP) in patients with systemic lupus erythematosus (SLE).

Membrane cofactor protein (MCP, CD46) is a cell surface complement regulatory protein which acts as a cofactor for the factor I-mediated cleavage of the activated complement components C3b/C4b. To evaluate the clinical usefulness of serum soluble CD46 as a marker of disease activity in patients with SLE, serum levels of sCD46 were measured by ELISA, using two MoAbs (M160 and M177), each of which recognized two different epitopes on CD46 molecule in SLE, other autoimmune diseases and healthy controls. Serum sCD46 levels in active SLE patients (30.5 +/- 14.1 ng/ml) were significantly higher than those of inactive SLE (5.8 +/- 7.1 ng/ml; P = 0.0003), rheumatoid arthritis (14.9 +/- 11.6 ng/ml; P = 0.0218), primary Sjögren's syndrome (12.3 +/- 11.6 ng/ml; P = 0.0039) and normal controls (7.3 +/- 3.6 ng/ml; P = 0.0005). The elevated serum sCD46 levels in active SLE patients significantly decreased from 30.5 +/- 14.1 ng/ml to 8.0 +/- 6.3 ng/ml after effective corticosteroid and immunosuppressant therapy (P = 0.018). Additionally, we found a significant negative association between increasing concentration of sCD46 and decreasing levels of CH50 in SLE (r = -0.598, P = 0.0009). These results suggest that sCD46 reflects in vivo activation of complement system and provides an additional useful serum parameter of active SLE.

C5b-9 membrane attack complex mediates endothelial cell apoptosis in experimental glomerulonephritis.

We studied the role of the C5b-9 membrane attack complex in two models of inflammatory glomerulonephritis (GN) initiated by acute glomerular endothelial injury in Piebold-viral-Glaxo (PVG) complement-sufficient rats (C+), C6-deficient rats (C6-), and rats systematically depleted of complement with cobra venom factor (CVF). GN was induced by performing a left nephrectomy and selectively perfusing the right kidney with either 1) the lectin concanavalin A (Con A) followed by complement-fixing anti-Con A (Con A GN) or 2) purified complement-fixing goat anti-rat glomerular endothelial cell (GEN) antibody [immune-mediated thrombotic microangiopathy (ITM)]. Comparable levels of GEN apoptosis were detected in C+ animals in both models. CVF administration reduced GEN apoptosis by 10- to 12-fold. GEN apoptosis was C5b-9 dependent because PVG C6- rats were protected from GEN loss. Furthermore, functional inhibition of the cell surface complement regulatory protein CD59 by renal perfusion with anti-CD59 antibody in ITM resulted in a 3.5-fold increase in GEN apoptosis. Last, in Con A GN, abrogation of GEN apoptosis preserved endothelial integrity and renal function. This study demonstrates the specific role of C5b-9 in the induction of GEN apoptosis in experimental inflammatory GN, a finding with implications for diseases associated with the presence of antiendothelial cell antibodies.

Expression of Human Complement Regulatory Protein CD46 Restricts Measles Virus Replication in Mouse Macrophages

Measles virus (MV) can infect mouse macrophages to cause a prolonged non-cytopathic infection that produces low levels of infectious virus for days. We have generated RAW264.7 mouse macrophages expressing human CD46, a cell surface complement regulatory protein that serves as a receptor for laboratory-adapted strains of MV. Laboratory-adapted MV strains efficiently enter the CD46-positive mouse macrophages to cause a cytopathic infection with extensive multinucleated cells and pseudopodia-like extensions. However, MV infection of mouse macrophages through CD46 is self-limiting. Both viral protein synthesis and infectious virus production are abruptly terminated after the second day of infection. This novel virus–cell interaction is seen only in mouse macrophages but not in mouse or hamster fibroblasts expressing human CD46. The possible role of CD46 in macrophage antiviral response restricting MV replication is discussed.

Molecular cloning of a pig homologue of membrane cofactor protein (CD46).

Organs of transgenic pigs that express human complement regulatory proteins are under assessment as an alternative to transplantation. A major barrier to the transplantation of pig organs is the hyperacute rejection caused by pre-existing antibodies and complement. Pig cells are very susceptible to human complement, presumably because pig cell-surface complement regulatory proteins are inefficient against it. Expression of human complement regulatory proteins, such as decay-accelerating factor and membrane cofactor proteins (MCP or CD46), by means of transgenes would confer resistance to human complement upon pig cells, thereby preventing hyperacute rejection. To express sufficient levels of human complement regulatory proteins at appropriate sites, regulatory elements of genes of pig membrane-bound complement regulatory proteins would be useful. To obtain their cDNAs, we transfected human cells with a pig cDNA library, selected cells by incubation with pig complement and rescued the plasmids. We cloned a cDNA for the pig homologue of MCP, pMCP. The cDNA encoded a predicted protein of 363 amino acids with 42% amino acid identity with human MCP. The pMCP consisted of four short consensus repeats, a Ser/Thr/Pro-rich domain, and transmembrane and cytoplasmic domains. Recombinant soluble pMCP that lacked transmembrane and cytoplasmic domains had factor I cofactor activity in C3b cleavage, indicating that it is functionally, as well as structurally homologous to MCP. FACS analysis with anti-pMCP mAb demonstrated that pMCP is expressed on all blood leukocytes, erythrocytes, and on endothelial and epithelial cell lines.

Soluble CD46 (membrane cofactor protein, MCP) in human reproductive tract fluids

CD46 (membrane cofactor protein, MCP) is a cell surface complement regulatory protein which may have an additional role in human sperm-egg interaction. A soluble form (sCD46) has also been detected in a number of biological fluids, most notably seminal plasma. The present study has employed a monoclonal antibody-based ELISA to assay sCD46 in reproductive tract fluids in normal and pathological conditions. Large amounts of sCD46 were detected in seminal plasma of both fertile and infertile men (combined mean, 4859 ng/ml). Vasectomized men had lower levels (mean, 2421 ng/ml), indicating contributory sources both before and after the vas deferens ligation site. Pre-colostrum also contained relatively high quantities (mean, 445 ng/ml), whereas breast milk (mean, 117 ng/ml), peritoneal fluid (mean, 154 ng/ml) and follicular fluid (mean, 107 ng/ml), as well as uterine (mean, 208 ng/ml), umbilical (mean, 166 ng/ml) and peripheral (mean, 206 ng/ml) blood plasma, had sCD46 levels within a comparable range. Amniotic fluid had low sCD46 concentrations (mean, 22 ng/ml). In endometriosis, peritoneal fluid levels of sCD46 were significantly raised (mean, 199 mg/ml). These results indicate distinctive fluid compartmentalisation of sCD46 consistent with a biological function in human reproductive tract fluids.

Expression of complement regulatory proteins on fetal blood cells in utero.

Since the fetus is semiallogenic to the mother, mechanisms have evolved to protect fetal tissue from the maternal immune response. Among these mechanisms is the expression of cell-surface complement regulatory proteins at the maternal-fetal interface. However, beginning in the third trimester, fetal blood cells are exposed to actively-transported IgG antibody. Thus, we speculated that fetal blood cells would require expression of one or more complement regulators by the early third trimester. Using flow cytometry and Western blots, we have demonstrated the presence of three important complement regulatory proteins in the circulating blood cells of human fetuses. These findings are consistent with the putative biological role of the cell-surface complement regulatory proteins.

Materno-fetal immunobiology in normal pregnancy and its possible failure in recurrent spontaneous abortion?

Many features contributing to the success of pregnancy in humans have been identified over the last 20 years. Trophoblast cells (which form the interface of fetal tissue with the mother) have specialized immunological features which may confer unique transplantation protection for the fetus throughout pregnancy. Both syncytiotrophoblast and cytotrophoblast cells do not express classical class I [human leucocyte antigen (HLA)-A or -B] or II (HLA-DP, -DQ or -DR) major histocompatibility complex (MHC) alloantigens, and the regulation of these cell surface glycoproteins appears to be at the transcriptional level. In contrast, extravillous cytotrophoblast cells express the non-classical class I MHC molecule HLA-G. One form of HLA-G (HLA-G1) is potentially capable of presenting a variety of peptide antigens to T cells. Alternatively, HLA-G may act as a cell surface class I MHC molecule, protecting cytotrophoblast from maternal MHC non-restricted natural killer (NK) cell attack; the expression of HLA-G by otherwise HLA-null cell transfectants has been shown to decrease their sensitivity to NK cell-mediated cytolysis. All fetal trophoblast populations throughout gestation express high levels of cell surface complement regulatory proteins, providing protection from complement-mediated damage at the materno-fetal interface. Analyses of trials with allogeneic leucocytes for the treatment of recurrent spontaneous abortion show little evidence of an improved success rate in immunized patients.

Diminished Expression of Cell-Surface Complement Regulatory Proteins in HIV-Infected Children and with HIV Infection of Peripheral Blood Mononuclear Cells in Vitro

Experimental data have established that HIV-infected lymphocytes activate the complement system. However, because mammalian lymphocytes possess a series of cell-surface complement regulators that inhibit amplification on autologous cells, complement-mediated destruction of host cells is usually inhibited. These studies were performed to examine whether alterations in the cell-surface complement regulatory proteins decay-accelerating factor (DAF, CD55) and membrane cofactor protein (MCP, CD46) may occur during HIV infection in vitro or in vivo. The physiologic significance of these alterations were assessed by radiolabeled chromium release experiments. We show that MCP fluorescent intensity is significantly lessened in HIV-infected children and that DAF intensity is similarly lessened in infected children with advanced disease. These findings could be duplicated with HIV infection of peripheral blood mononuclear cells in vitro.

Polymorphism of the human CD46 gene in normal individuals and in recurrent spontaneous abortion

Restriction fragment length polymorphism analysis of the human CD46 gene, which encodes a cell surface complement regulatory protein, has been performed using a 1.5-kb cDNA probe containing a complete CD46 coding sequence. The sum of the invariant band sizes indicates that this gene encompasses at least 35-kb genomic DNA. Polymorphisms were detected in normal individuals with both HindIII and EcoRI. Several of the polymorphic bands occurred significantly less frequently in recurrent spontaneous abortion individuals. Northern blot analysis has shown the predominant RNA transcript size to be 4.2 kb in trophoblast-derived cell lines and peripheral blood mononuclear cells, indicating these transcripts may contain an unusually long 3′-untranslated region.

Application of Protein A-Rosette Assay for Screening of Monoclonal Antibodies to Human Complement Regulatory Proteins

Mice were immunized with purified membrane cofactor protein (MCP) and its monoclonal antibodies were screened by protein A(PA)-rosette assay. In this assay, the culture supernatants of hybridoma cells were layered over fixed MCP-bearing cells, and after washing, PA-coated sheep erythrocytes were applied as an indicator to these MCP-bearing cells. No purified antigen was therefore required throughout the screening. More than 300 of the supernatants harvested were successfully examined within 6 h. Each resultant antibody consisted of a single subclass of IgG, and reacted only with MCP in both transblotted and surface-labeled materials. The sensitivity of this assay was then assessed with these purified antibodies. As little as 0.5 micrograms of IgG1 or 0.01 micrograms of IgG2a was found to be detectable with more than 30% rosette formation. There were variations among cell lines in the sensitivity to the PA-rosette assay and the sensitivity did not correlate with the quantity of MCP surface expression in any of the cell lines. K562 gave the lowest background (nonspecific rosette formation) and the best specificity for anti-MCP of the 20 MCP-positive cell lines tested. Cell lines suitable for the detection of monoclonal anti-decay-accelerating factor and anti-C3b/C4b receptor were also examined and CCRF-SB and HSB2, and peripheral blood granulocytes, were found to be proper cell lines for screening the decay-accelerating factor and C3b/C4b receptor, respectively. Clones for anti C3b/C4b receptor were successfully obtained using granulocytes by the PA-rosette assay. This method needs no purified antigen and facilitates the rapid screening and purification of positive clones against cell-surface complement regulatory proteins.