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Complement receptor of immunoglobulin superfamily (CRIg) is expressed on liver macrophages and directly binds complement component C3b or Gram-positive bacteria to mediate phagocytosis. CRIg plays important roles in several immune-mediated diseases, but it is not clear how its pathogen recognition and phagocytic functions maintain homeostasis and prevent disease. We previously associated cytolysin-positive Enterococcus faecalis with severity of alcohol-related liver disease. Here, we demonstrate that CRIg is reduced in liver tissues from patients with alcohol-related liver disease. CRIg-deficient mice developed more severe ethanol-induced liver disease than wild-type mice; disease severity was reduced with loss of toll-like receptor 2. CRIg-deficient mice were less efficient than wild-type mice at clearing Gram-positive bacteria such as Enterococcus faecalis that had translocated from gut to liver. Administration of the soluble extracellular domain CRIg-Ig protein protected mice from ethanol-induced steatohepatitis. Our findings indicate that ethanol impairs hepatic clearance of translocated pathobionts, via decreased hepatic CRIg, which facilitates progression of liver disease., (© 2021. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Human complement receptor 1 (HuCR1) is a pivotal regulator of complement activity, acting on all three complement pathways as a membrane-bound receptor of C3b/C4b, C3/C5 convertase decay accelerator, and cofactor for factor I-mediated cleavage of C3b and C4b. In this study, we sought to identify a minimal soluble fragment of HuCR1, which retains the complement regulatory activity of the wildtype protein. To this end, we generated recombinant, soluble, and truncated versions of HuCR1 and compared their ability to inhibit complement activation in vitro using multiple assays. A soluble form of HuCR1, truncated at amino acid 1392 and designated CSL040, was found to be a more potent inhibitor than all other truncation variants tested. CSL040 retained its affinity to both C3b and C4b as well as its cleavage and decay acceleration activity and was found to be stable under a range of buffer conditions. Pharmacokinetic studies in mice demonstrated that the level of sialylation is a major determinant of CSL040 clearance in vivo. CSL040 also showed an improved pharmacokinetic profile compared with the full extracellular domain of HuCR1. The in vivo effects of CSL040 on acute complement-mediated kidney damage were tested in an attenuated passive antiglomerular basement membrane antibody-induced glomerulonephritis model. In this model, CSL040 at 20 and 60 mg/kg significantly attenuated kidney damage at 24 h, with significant reductions in cellular infiltrates and urine albumin, consistent with protection from kidney damage. CSL040 thus represents a potential therapeutic candidate for the treatment of complement-mediated disorders., Competing Interests: Conflict of interest S. W., H. C., A. B. M., T. R., and M. P. H. are listed as inventors on International Patent Publication number WO2019/218009. All authors with the exception of A. G. N., G. A. P., G. M.- R., M. M., and M. S. are CSL shareholders., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Nanodrug carrier will eventually enter the blood when intravenously injected or in other ways. Meanwhile, a series of toxic effects were caused to the body with the formation of nanoparticle protein corona. In our studies, we try to reveal the recognition mechanism of nanoparticle protein corona by monocyte and the damage effect on immune cells by activated complement of hydroxyapatite nanoparticles (HAP-NPs) and silicon dioxide nanoparticles (SiO 2 -NPs). So expressions of TLR4/CR1/CR were analyzed by flow cytometry (FCM) in order to illuminate the recognition mechanism of nanoparticle protein corona by monocyte. And the expression of ROS, cytokines, adhesion molecules, and arachidonic acid was measured when THP-1 and HUVECs were stimulated by NP-activated complement. The results showed that HAP-NPs can be recognized by the opsonin receptor (iC3b/CR3) model, while plasma protein, opsonin receptor, and Toll-like receptors are all likely launch cell recognition of SiO 2 -NPs. And it was considerate that NP-activated complement can damage THP-1 and HUVECs, including oxidative stress, inflammation, and increased vascular permeability. So the surface of nanodrug carrier can be modified to avoid being clear and reduce the efficacy according to the three receptors (TLR4/CR1/CR3)., Competing Interests: The authors declare that they have no conflict of interest., (Copyright © 2020 Tingting Ding and Jiao Sun.)

BACKGROUND The aim of this study was to determine the relationship between the expression of CD35 and CD64 from white blood cells (neutrophil, monocytes, and lymphocytes) and acute infectious diseases in children. MATERIAL AND METHODS The blood samples were collected from 104 children with infections (42 viral infections and 62 bacterial infections). Blood samples were stained with CD45-PC5, CD35-FITC, and CD64-PE, and the fluorescence intensities were measured by flow cytometer, and then the ratio of CD35 to CD64 was calculated. RESULTS The ratio of CD64/CD35 on neutrophils (NCD35/NCD64) was significantly different between the bacterial group, the virus group, and the healthy control group. According to receiver operating characteristic (ROC) analysis, a cutoff value of 7.256 (sensitivity: 90.0%, specificity: 93.7%) was determined for the NCD35/NCD64 ratio. CONCLUSIONS This study shows that NCD35/NCD64 is helpful in the differential diagnosis of acute viral infection and bacterial infection in children.

Type 1 diabetes mellitus is an autoimmune metabolic disorder characterized by chronic hyperglycemia, the presence of autoreactive T and B cells and autoantibodies against self-antigens. A membrane-bound enzyme on the pancreatic beta-cells, glutamic acid decarboxylase 65 (GAD65), is one of the main autoantigens in type 1 diabetes. Autoantibodies against GAD65 are potentially involved in beta-cell destruction and decline of pancreatic functions. The human complement receptor type 1 (CD35) on B and T lymphocytes has a suppressive activity on these cells. We hypothesized that it may be possible to eliminate GAD65-specific B cells from type 1 diabetes patients by using chimeric molecules, containing an anti-CD35 antibody, coupled to peptides resembling GAD65 B/T epitopes. These molecules are expected to selectively bind the anti-GAD65 specific B cells by the co-cross-linking of the immunoglobulin receptor and CD35 and to deliver a suppressive signal. Two synthetic peptides derived from GAD65 protein (GAD65 epitopes) and anti-CD35 monoclonal antibody were used for the construction of two chimeras. The immunomodulatory activity of the engineered antibodies was tested in vitro using peripheral blood mononuclear cells (PBMCs) from type 1 diabetes patients. A reduction in the number of anti-GAD65 IgG antibody-secreting plasma cells and increased percentage of apoptotic B lymphocytes was observed after treatment of these PBMCs with the engineered antibodies. The constructed chimeric molecules are able to selectively modulate the activity of GAD65-specific B lymphocytes and the production of anti-GAD65 IgG autoantibodies by co-cross-linking of the inhibitory CD35 and the B cell antigen receptor (BCR). This treatment presents a possible way to alter the autoimmune nature of these cells., (© 2019 British Society for Immunology.)

Studies conducted in animal models have suggested that membrane complement regulatory proteins play an important role in the pathophysiology of coronary artery disease (CAD). In this study, a total of 100 individuals, with stable CAD and 100 healthy controls, both groups predominantly male, were recruited. We evaluated the plasma levels of complement regulatory proteins (Cregs) CD35, CD46, CD55, and CD59 and their surface expression on granulocytes, lymphocytes, and monocytes by flow cytometry. The mRNA expression of these Cregs in total leukocytes was determined by quantitative PCR. The soluble forms of Cregs, C3c, Mannose binding protein-associated serine protease 2 (MASP-2), Platelet activating factor-acetyl hydrolase (PAF-AH), and inflammatory cytokines were quantified by ELISA. High plasma levels of C3c, indicative of complement activation, in addition to significantly low levels of Cregs, were observed in CAD patients. A significantly lower expression of CD46 and CD55 on the surface of lymphocytes, monocytes, and granulocytes and higher surface expression of CD35 and CD59 on granulocytes ( p < 0.0001) was seen in CAD patients as compared to healthy donors. The high expression of CD59 on granulocytes positively correlated with the severity of disease and may serve as a potential marker of disease progression in CAD.

RBC alloimmunization represents a significant immunological challenge for patients requiring lifelong transfusion support. The majority of clinically relevant non-ABO(H) blood group antigens have been thought to drive antibody formation through T cell-dependent immune pathways. Thus, we initially sought to define the role of CD4+ T cells in formation of alloantibodies to KEL, one of the leading causes of hemolytic transfusion reactions. Unexpectedly, our findings demonstrated that KEL RBCs actually possess the ability to induce antibody formation independent of CD4+ T cells or complement component 3 (C3), two common regulators of antibody formation. However, despite the ability of KEL RBCs to induce anti-KEL antibodies in the absence of complement, removal of C3 or complement receptors 1 and 2 (CR1/2) rendered recipients completely reliant on CD4+ T cells for IgG anti-KEL antibody formation. Together, these findings suggest that C3 may serve as a novel molecular switch that regulates the type of immunological pathway engaged following RBC transfusion.

Complement receptor type 1 (CR1) is a multi modular membrane receptor composed of 30 homologous complement control protein modules (CCP) organized in four different functional regions called long homologous repeats (LHR A, B, C, and D). CR1 is a receptor for complement-opsonins C3b and C4b and specifically interacts through pairs of CCP modules located in LHR A, B, and C. Defense collagens such as mannose-binding lectin (MBL), ficolin-2, and C1q also act as opsonins and are involved in immune clearance through binding to the LHR-D region of CR1. Our previous results using deletion variants of CR1 mapped the interaction site for MBL and ficolin-2 on CCP24-25. The present work aimed at deciphering the interaction of C1q with CR1 using new CR1 variants concentrated around CCP24-25. CR1 bimodular fragment CCP24-25 and CR1 CCP22-30 deleted from CCP24-25 produced in eukaryotic cells enabled to highlight that the interaction site for both MBL and C1q is located on the same pair of modules CCP24-25. C1q binding to CR1 shares with MBL a main common interaction site on the collagen stalks but also subsidiary sites most probably located on C1q globular heads, contrarily to MBL.

The B7 family-related protein, V-set and Ig domain (VSIG4) / Z39Ig / complement receptor immunoglobulin (CRIg), is a new player in the regulation of immunity to infection and inflammation. The unique features of this receptor as compared with classical complement receptors, CR3 and CR4, have heralded the emergence of new concepts in the regulation of innate and adaptive immunity. Its selective expression in tissue macrophages and dendritic cells has been considered of importance in host defence and in maintaining tolerance against self-antigens. Although a major receptor for phagocytosis of complement opsonised bacteria, its array of emerging functions which incorporates the immune suppressive and anti-inflammatory action of the receptor have now been realised. Accumulating evidence from mouse experimental models indicates a potential role for CRIg in protection against bacterial infection and inflammatory diseases, such as rheumatoid arthritis, type 1 diabetes and systemic lupus erythematosus, and also in promotion of tumour growth. CRIg expression can be considered as a control point in these diseases, through which inflammatory mediators, including cytokines, act. The ability of CRIg to suppress cytotoxic T cell proliferation and function may underlie its promotion of cancer growth. Thus, the unique properties of this receptor open up new avenues for understanding of the pathways that regulate inflammation during infection, autoimmunity and cancer with the potential for new drug targets to be identified. While some complement receptors may be differently expressed in mice and humans, as well as displaying different properties, mouse CRIg has a structure and function similar to the human receptor, suggesting that extrapolation to human diseases is appropriate. Furthermore, there is emerging evidence in human conditions that CRIg may be a valuable biomarker in infection and immunity, inflammatory conditions and cancer prognosis.

The spread of methicillin-resistant Staphylococcus aureus (MRSA) is a critical health issue that has drawn greater attention to the potential use of immunotherapy. Toll-like receptor 2 (TLR2), a pattern recognition receptor, is an essential component in host innate defense system against S. aureus infection. However, little is known about the innate immune response, specifically TLR2 activation, against MRSA infection. Here, we evaluate the protective effect and the mechanism of MRSA murine pneumonia after pretreatment with Pam3CSK4, a TLR2 agonist. We found that the MRSA-pneumonia mouse model, pretreated with Pam3CSK4, had reduced bacteria and mortality in comparison to control mice. As well, lower protein and mRNA levels of TNF-α, IL-1β and IL-6 were observed in lungs and bronchus of the Pam3CSK4 pretreatment group. Conversely, expression of anti-inflammatory cytokine IL-10, but not TGF-β, increased in Pam3CSK4-pretreated mice. Our additional studies showed that CXCL-2 and CXCL1, which are necessary for neutrophil recruitment, were less evident in the Pam3CSK4-pretreated group compared to control group, whereas the expression of Fcγ receptors (FcγⅠ/Ⅲ) and complement receptors (CR1/3) increased in murine lungs. Furthermore, we found that increased survival and improved bacterial clearance were not a result of higher levels of neutrophil infiltration, but rather a result of enhanced phagocytosis and bactericidal activity of neutrophils in vitro and in vivo as well as increased robust oxidative activity and release of lactoferrin. Our cumulative findings suggest that Pam3CSK4 could be a novel immunotherapeutic candidate against MRSA pneumonia.

Complement receptors (CRs) play an integral role in innate immunity and also function to initiate and shape the adaptive immune response. Our earlier results showed that complement receptor type 1 (CR1, CD35) is a potent inhibitor of the B cell receptor- (BCR-) induced functions of human B lymphocytes. Here we show that this inhibition occurs already at the initial steps of B cell activation since ligation of CR1 reduces the BCR-induced phosphorylation of key signaling molecules such as Syk and mitogen activated protein kinases (MAPKs). Furthermore, our data give evidence that although B lymphocytes of active systemic lupus erythematosus (SLE) patients express lower level of CR1, the inhibitory capacity of this complement receptor is still maintained and its ligand-induced clustering results in significant inhibition of the main B cell functions, similar to that found in the case of healthy individuals. Since we have found that reduced CR1 expression of SLE patients does not affect the inhibitory capacity of the receptor, our results further support the therapeutical potential of CD35 targeting the decrease of B cell activation and autoantibody production in autoimmune patients., Competing Interests: The authors declare no financial or commercial conflict of interests.

In this paper we have extended our earlier studies of the action of increasing Factor I concentration on complement activation by using a soluble activator, lipopolysaccharide (LPS) endotoxin, and using human erythrocytes as a source of CR1 - the co-factor needed for the final clip of iC3b to C3dg by Factor I. Using this more physiological system, the results show that we can predict that a quite modest increase in Factor I concentration - 22 µg/ml of extra Factor I - will convert the activity of the highest risk sera to those of the lowest risk. Preliminary experiments have been performed with erythrocytes allotyped for CR1 number. While we have not been able to perform an adequate study of their co-factor activities in our assays, preliminary experiments suggest that when Factor I levels are increased the difference produced by different allotypes of red cells is largely overcome. This suggests that in patients with paroxysmal nocturnal haemoglobinuria (PNH) treated with eculizumab, additional treatment with Factor I may be very useful in reducing the need for blood transfusion. We have also explored the age-related allele frequency for the two polymorphisms of Factor H and the polymorphism of C3. In our population, unlike the 1975 study, we found no age variation in the allele frequency in these polymorphisms. This may, however, reflect that the Cambridge BioResource volunteers do not include many very young or very elderly patients, and in general comprise a population not greatly at risk of death from infectious disease., (© 2015 British Society for Immunology.)

In the present study, we obtain a mouse anti-porcine complement receptor type 1 (CR1)-like monoclonal antibody (McAb) and use this McAb to verify the existence of CR1-like protein on porcine erythrocytes. Our results confirm that CR1-like protein is localized on the surface of porcine erythrocytes. Mouse immunoglobulin G inhibited the binding of serum-opsonized green fluorescent protein-expressing Escherichia coli to porcine erythrocytes. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis indicates that CR1-like McAb reacts with biochemically-purified porcine erythrocyte membrane fractions, with a clear band at 135 kDa to 140 kDa. We postulate that the 135 kDa to 140 kDa membrane protein is the equivalent of the porcine erythrocyte CR1-like protein.

Follicular dendritic cells (FDCs) regulate B cell function and development of high affinity antibody responses but little is known about their biology. FDCs associate in intricate cellular networks within secondary lymphoid organs. In vitro and ex vivo methods, therefore, allow only limited understanding of the genuine immunobiology of FDCs in their native habitat. Herein, we used various multicolor fate mapping systems to investigate the ontogeny and dynamics of lymph node (LN) FDCs in situ. We show that LN FDC networks arise from the clonal expansion and differentiation of marginal reticular cells (MRCs), a population of lymphoid stromal cells lining the LN subcapsular sinus. We further demonstrate that during an immune response, FDCs accumulate in germinal centers and that neither the recruitment of circulating progenitors nor the division of local mature FDCs significantly contributes to this accumulation. Rather, we provide evidence that newly generated FDCs also arise from the proliferation and differentiation of MRCs, thus unraveling a critical function of this poorly defined stromal cell population., (© 2014 Jarjour et al.)

The pathogenesis of malaria, an insect-borne disease that takes millions of lives every year, is still not fully understood. Complement receptor 1 (CR1) has been described as a receptor for Plasmodium falciparum, which causes cerebral malaria in humans. We investigated the role of CR1 in an experimental model of cerebral malaria. Transgenic mice expressing human CR1 (hCR1(+)) on erythrocytes were infected with Plasmodium berghei ANKA and developed cerebral malaria. No difference in survival was observed in hCR1(+) mice compared to wild-type mice following infection with P. berghei ANKA; however, hCR1 detection was significantly diminished on erythrocytes between days 7 and 10 postinfection. hCR1 levels returned to baseline by day 17 postinfection in surviving animals. Immunoblot assays revealed that total erythrocyte hCR1 levels were diminished, confirming that immune complexes in association with erythrocyte hCR1 were likely removed from erythrocytes in vivo by clearance following immune adherence. Decreases in hCR1 were completely dependent on C3 expression, as mice treated with cobra venom factor (which consumes and depletes C3) retained hCR1 on erythrocytes during C3 depletion through day 7; erythrocyte hCR1 decreases were observed only when C3 levels recovered on day 9. B-cell-deficient mice exhibit a marked increase in survival following infection with P. berghei ANKA, which suggests that immune complexes play a central role in the pathogenesis of experimental cerebral malaria. Together, our findings highlight the importance of complement and immune complexes in experimental cerebral malaria. IMPORTANCE Cerebral malaria is a deadly complication of infection with Plasmodium falciparum. Despite its high prevalence, relatively little is understood about its pathogenesis. We have determined that immune complexes are generated and deposited on erythrocytes specifically expressing human complement receptor 1 in a mouse model of cerebral malaria. We also provide evidence demonstrating the importance of immunoglobulins in the pathogenesis of cerebral malaria in mice. These findings may have important implications in human cerebral malaria.

While the immunotoxicity of endosulfan has been studied, little is known about its influence on immune function associated with erythrocytes (RBC). The aim of this study was to investigate the possible effects of endosulfan, and any possible mitigation by testosterone propionate (TP), on erythrocyte immune function in a mouse model. To this end, rosette formation [as erythrocyte C3b receptor(E-C3bR) and erythrocyte immune complexes (E-IC)], as well as measures of the erythrocyte C3b receptor rosette-forming enhancing rate (RFER; reflecting immunoenhancing factor activity) and C3b receptor rosette-forming inhibitory rate (RFIR; reflecting immunosuppressive factor activity) were performed. The effects of RBC on regulating NK cell function or T-cell adherence were also analyzed. Lastly, to begin to assess potential mechanisms by which endosulfan could impact on the measured endpoints, CD35, CD58, and CD59 expression on RBC was evaluated; expression/mRNA levels of complement receptor I-related gene/protein y (Crry) on cells/splenic tissues was also assessed. The data show that E-C3bR rosette ratios decreased, and those of E-IC increased, due to endosulfan treatment. In these hosts, RFER (i.e., immunoenhancing factor in plasma) was decreased, but RFIR (i.e., immunosuppressive factor) was unchanged.There were no clear effects from endosulfan on RBC regulatory function against NK or T-cells. Lastly, Crry mRNA levels in tissues/cells from these mice were significantly decreased; however, CD59 and CD58 expression levels were unaffected. The data also show that TP co-treatment reversed or mitigated effects of endosulfan on each endpoint, in part, by two possible mechanisms; the TP may be increasing the activity of the innate immune enhancing factor, or, an anti-oxidant effect of TP might help to protect membrane structures and increase Crry stability on the RBC.

Specific immunotherapy (SIT) is the only specific remedy for the treatment of allergic diseases currently. B cells are important immune cells in the immunity. The role of B cells in immune regulatory activities has not been fully understood yet. This study aims to elucidate the role of the thrombospondin (TSP)1-producing B cells in the immune regulatory role of SIT. The results showed that after SIT, the frequency of CD35(+) B cells was increased in the intestine of mice with food allergy. The CD35(+) B cells expressed TSP1 after exposure to specific antigens. Co-culture with the TSP1-producing CD35(+) B cells decreased the levels of CD80/CD86 in dendritic cells; the cells convert naïve CD4(+) T cells to regulatory T cells to inhibit allergic inflammation in the intestine.

Humans and other higher primates are unique among mammals in using complement receptor 1 (CR1, CD35) on red blood cells (RBC) to ligate complement-tagged inflammatory particles (immune complexes, apoptotic/necrotic debris, and microbes) in the circulation for quiet transport to the sinusoids of spleen and liver where resident macrophages remove the particles, but allow the RBC to return unharmed to the circulation. This process is called immune-adherence clearance. In this study we found using luminometric- and fluorescence-based methods that ligation of CR1 on human RBC promotes ATP release. Our data show that CR1-mediated ATP release does not depend on Ca(2+) or enzymes previously shown to mediate an increase in membrane deformability promoted by CR1 ligation. Furthermore, ATP release following CR1 ligation increases the mobility of the lipid fraction of RBC membranes, which in turn facilitates CR1 clustering, and thereby enhances the binding avidity of complement-opsonized particles to the RBC CR1. Finally, we have found that RBC-derived ATP has a stimulatory effect on phagocytosis of immune-adherent immune complexes.

The involvement of B cells, complement activation and subsequent immune complex deposition has all been implicated in the pathogenesis of rheumatoid arthritis (RA). Although the reduced expression of complement receptor 1 (CR1, CD35) and 2 (CR2, CD21) on the B cells of RA patients has been known for a long time, their exact role in B-cell tolerance and autoimmunity is not yet fully understood. To get a deeper insight into the possible mechanisms, we studied the expression and function of CR1 and CR2 on various subsets of B cells of healthy donors and RA patients at various stages of the disease by FACS analysis, (3)H-thymidine incorporation and ELISA. We found that CD19(+)CD27(-) naive B cells up-regulate the expression of the inhibitory CR1 during differentiation to CD19(+)CD27(+) memory B cells both in healthy donors and in RA patients, whereas the expression of the activatory CR2 is down-regulated. This clearly demonstrates that the expression of these two antagonistic complement receptors is regulated differentially during the development of human B cells, a phenomenon which may influence the maintenance of peripheral B-cell tolerance. Our functional studies show that after clustering CR1 both by its natural ligand and To5 mAb, the inhibitory function of CD35 is maintained in RA patients, despite its significantly reduced expression compared with healthy individuals. Besides blocking B-cell receptor-induced proliferation, CR1 inhibits the differentiation of B cells to plasmablasts and their immunoglobulin production. Since the reduced expression of CR1 in RA patients does not affect its inhibitory function, this receptor might serve as a new target for therapeutical interventions.

The purpose of this research was to investigate if Vitamin E could decrease the toxic-effects of endosulfan on erythrocyte immunity and its regulating mechanism. The levels of endosulfan and Vitamin E were (in mg/kg/d), respectively, 0 and 0 (control group), 0.8 and 0 (endosulfan-only group), 0.8 and 100 (experimental group). The results showed that Vitamin E inhibited endosulfan-induced decreases in rosette ratios of erythrocyte C(3)b receptor and increases in rosette ratios of erythrocyte immune complex. Vitamin E reversed the decline tendency of erythrocytes to regulate T-lymphocyte activity and the increase tendency of erythrocyte immunosuppressive factor activity in plasma induced by endosulfan. Further, Vitamin E alleviated the decreases of CD35 mRNA levels in spleen and CD35 expression on B-lymphocyte surfaces, antagonized a decline in Crry mRNA levels. Lastly, Vitamin E reversed induced decreases in total anti-oxidation capability and increases in malondi-aldehyde and free radical levels in spleen caused by endosulfan. The results suggested that Vitamin E relieved endosulfan-induced effects on erythrocytes immunity, reversed changes in expression of erythrocyte immune factors, and antagonized oxidative stress. Vitamin E could stabilize the expression of Crry receptor by inhibiting oxidative stress, and thereby reverse the decrease of erythrocyte immunity caused by endosulfan.

Mannan-binding lectin (MBL), a lectin homologous to C1q, greatly facilitates C3/C4-mediated opsonophagocytosis of Candida albicans (C. albicans) by human neutrophils, and has the capacity to bind to CR1 (CD35) expressed on circulating neutrophils. The intracellular pool of neutrophil Dectin-1 plays a critical role in stimulating the reactive oxygen species (ROS) generation through recognition of β-1,3-glucan component of phagocytized zymosan or yeasts. However, little is known about whether MBL can mediate the opsonophagocytosis of Candida albicans by neutrophils independent of complement activation, and whether MBL-mediated opsonophagocytosis influence the intracellular expression of Dectin-1 and ROS production. Here we showed that the inhibited phagocytic efficiency of neutrophils as a result of blockage of Dectin-1 was compensated by exogenous MBL alone in a dose-dependent manner. Furthermore, the expressions of Dectin-1 at mRNA and intracellular protein levels were significantly up-regulated in neutrophils stimulated by MBL-pre-incubated C. albicans, while the expression of surface Dectin-1 remained almost unchanged. Nevertheless, the stimulated ROS production in neutrophils was partly and irreversibly inhibited by blockage of Dectin-1 in the presence of exogenous MBL. Confocal microscopy examination showed that intracellular Dectin-1 was recruited and co-distributed with ROS on the surface of some phagocytized yeasts. The β-1,3-glucanase digestion test further suggested that the specific recognition and binding site of human Dectin-1 is just the β-1,3-glucan moiety on the cell wall of C. albicans. These data demonstrate that MBL has an ability to mediate the opsonophagocytosis of Candida albicans by human neutrophils independent of complement activation, which is coupled with intracellular Dectin-1-triggered ROS production.

Complement receptor 1 (CR1) levels have been associated with malarial susceptibility and/or severity of the disease in different population groups, and CR1 is a receptor for Plasmodium falciparum. In this study, multiple CR1 single-nucleotide polymorphisms (SNPs) showed strong evidence of population differentiation between Sardinian and other European ethnic groups. Cross population algorithms comparing haplotype structure and differences in haplotype and allele frequency distribution provided additional support for natural selection of CR1 in Sardinia. The predominant Sardinian CR1 haplotype included SNPs that are associated with decreased CR1 levels in Europeans and other population groups. Previous studies have shown that the SNPs within the dominant Sardinian haplotype have a significantly higher frequency in a malaria endemic compared with non-endemic regions in India. Together with the historical evidence of the prevalence of malaria in Sardinia, these data support the role of malaria leading to positive selection of this CR1 haplotype in Sardinia.

Tigecycline achieves high intracellular concentrations in polymorphonuclear leukocytes (PMNs). To evaluate the effects of tigecycline on human PMNs, PMNs were incubated with tigecycline dilutions (0.1 to 100 mg L-1). Phagocytosis-associated PMN Fcγ- and complement receptors as well as phagocytosis and oxidative burst induced by Staphylococcus aureus were measured by flow cytometry. Incubation with tigecycline caused small but significant decreases in the density of complement receptors CD11b and CD35 (all concentrations) and Fcγ receptors CD16 and CD32 (high concentrations), but not in the percentages of receptor-bearing cells, except for small reductions in the proportions of CD16 positive cells at high concentrations. Tigecycline had no effect on phagocytosis or oxidative burst induced by S. aureus. Tigecycline was thus associated with decreased density of PMN complement and (at high concentrations) Fcγ receptors. Although statistically significant, the differences were small and did not influence the PMN function as measured by phagocytosis and oxidative burst.

Background & Objectives: Severe anaemia in Plasmodium falciparum (Pf) associated malaria is a leading cause of death despite low levels of parasitaemia. In an effort to understand the pathogenesis of anaemia we studied expression level of RBC complement regulatory proteins, CR1 (CD35), CD55 and CD59 with haemoglobin status in a group of malaria cases from Assam, Goa and Chennai, and in healthy controls., Methods: Flowcytometry was used to study expression of CR1, CD55 and CD59 in 50 Pf cases and 30 normal healthy volunteers. Giemsa stained thick and thin blood films were used for microscopic detection and identification of malarial parasites and parasite count., Results: No correlation was found between degree of expression of RBC surface receptors CR1, CD55 and CD59 with haemoglobin level. However, expression of CD55 was less in malaria cases than in healthy controls., Interpretation & Conclusions: The present findings indicate that malaria infection changes the expression profile of complement regulatory protein CD55 irrespective of severity status of anaemia. Further studies are needed to explore the pathophysiology of anaemia in malaria cases in Assam where expression of RBC complement receptors appears to be low even in normal healthy population.

Lymph nodes (LN) are highly organized and have characteristic compartments. Destruction of these compartments leads to an inability to fulfil their immunological function. However, it is not yet clearly understood which mechanisms are involved in the development and maintenance of this organization. After transplantation of LN into the mesentery, the LN regenerate to fully functional LN. In this study, the question was addressed, how stromal cells in the B-cell follicles (follicular dendritic cells), which were identified by CD21/CD35, and stromal cells in the T-cell area (gp38+ cells) are involved via chemokine signalling. The gp38+ cells and CD21/CD35+ cells were detected in the transplanted LN (EGFP, plt/plt and CXCR5(-/-) mice) over a period of 8 weeks to analyse their competence to reconstruct the compartmental organization. The presence of gp38+ cells was stable during regeneration and these cells reconstructed the T-cell area within 4 weeks. After transplantation of plt/plt LN CCL19/CCL21 expression was observed leading to partial restoration of the T-cell area. In contrast, there were changes in the presence and morphology of CD21/CD35+ cells within the B-cell area during reconstruction, which was dependent on the presence of B cells and CXCL13/CXCR5 signalling. Hence, CD21/CD35+ cells and gp38+ cells are involved in the establishment of the compartmental organization of lymph nodes but using different ways to recruit lymphocytes via chemokine signalling., (© 2011 The Authors. Immunology © 2011 Blackwell Publishing Ltd.)

As a crucial element of innate immunity, the complement cascade becomes activated after severe trauma. Regulation of the complement cascade and protection against complement-mediated tissue destruction is provided by a selection of soluble and membrane-bound complement regulatory proteins (CRegs). To date, the leukocyte expression profile of CRegs in multiple injured patients is unknown. In the present study, expression of CRegs and the C5a receptor (CD88) was analyzed on neutrophils, monocytes, and lymphocytes by flow cytometry. Whole blood samples were obtained from healthy volunteers (n = 16) or multiple injured patients (n = 12) on admission in the emergency department and 4, 12, 24, 120, and 240 h after trauma. The content of CRegs and CD88 on leukocytes was significantly altered posttrauma: CD55 (decay accelerating factor) displayed a time-dependent, elevated expression pattern on neutrophils and monocytes, but not on lymphocytes. CD59 (membrane attack complex inhibitor) expression was significantly increased on neutrophils and monocytes at the time of admission and after 5 to 10 days in lymphocytes. CD46 (membrane cofactor protein) was significantly down-regulated in all three cell types posttrauma. CD35 (complement receptor 1) expression on neutrophils was initially decreased, whereas monocytes presented a significant increase in CD35 expression. CD35 on lymphocyte remained unchanged throughout the observation period. CD88 expression was considerably reduced on leukocytes between 0 and 240 h after injury. CD59, CD46, and CD88 expression values on neutrophils reversely correlated with severity of injury. In summary, expression profiles of CRegs and CD88 on leukocytes are specifically altered after polytrauma in humans, indicating a trauma-induced "complementopathy."

The absence of complement receptor 1 (CR1) related gene/protein y (Crry) leads to embryonic lethality as a result of unrestricted complement activation and concomitant neutrophil infiltration. Here we used Crry(-/-)C3(+/-) mice to investigate the role of Crry in the pathogenesis of immune complex glomerulonephritis (GN). After 3 weeks of immunization with horse spleen apoferritin, six of nine Crry(-/-) C3(+/-) mice and none of the six control C3(+/-) mice developed proliferative GN (P = 0.010). After 5 weeks of immunization, GN scores in Crry(-/-) C3(+/-) mice were 0.67 +/- 0.22 mean +/- standard error of the mean (SEM), compared with 0.32 +/- 0.16 in C3(+/-) mice. Glomerular hypercellularity was attributable to neutrophil infiltration in mice with GN (1.7 +/- 0.3/glomerulus) compared with those without GN (0.4 +/- 0.1/glomerulus) (P = 0.001). Absent staining for alpha-smooth muscle actin and proliferating cell nuclear antigen suggested that mesangial cell proliferation did not play a significant role in this model. Serum C3 levels in Crry(-/-) C3(+/-) mice were approximately 20% and 30% those of wild-type mice and C3(+/-) mice, respectively. To determine whether this acquired hypocomplementaemia was relevant to this GN model system, Crry(-/-) C3(+/-) mouse kidneys were transplanted into wild-type mice followed by immunization with apoferritin for 1 or 2 weeks. Surprisingly, none of the Crry(-/-) C3(+/-) mouse kidneys developed GN at these early time-points, indicating that increasing circulating C3 levels several-fold did not increase susceptibility to GN. Renal expression of decay-accelerating factor was not different among any of the groups studied. Thus, our data indicate that mesangial cell Crry limits complement activation and subsequent neutrophil recruitment in the setting of local immune complex deposition.

Background: Severe anemia due to Plasmodium falciparum malaria is a major cause of mortality among young children in western Kenya. The factors that lead to the age-specific incidence of this anemia are unknown. Previous studies have shown an age-related expression of red cell complement regulatory proteins, which protect erythrocytes from autologous complement attack and destruction. Our primary objective was to determine whether in a malaria-endemic area red cells with low levels of complement regulatory proteins are at increased risk for complement (C3b) deposition in vivo. Secondarily, we studied the relationship between red cell complement regulatory protein levels and hemoglobin levels., Methods: Three hundred and forty-two life-long residents of a malaria-holoendemic region of western Kenya were enrolled in a cross-sectional study and stratified by age. We measured red cell C3b, CR1, CD55, and immune complex binding capacity by flow cytometry. Individuals who were positive for malaria were treated and blood was collected when they were free of parasitemia. Analysis of variance was used to identify independent variables associated with the %C3b-positive red cells and the hemoglobin level., Results: Individuals between the ages of 6 and 36 months had the lowest red cell CR1, highest %C3b-positive red cells, and highest parasite density. Malaria prevalence also reached its peak within this age group. Among children 6 to

Plasmodium falciparum malaria causes 1-2 million deaths per year. Most deaths occur as a result of complications such as severe anemia and cerebral malaria (CM) (coma). Red cells of children with severe malaria-associated anemia (SMA) have acquired deficiencies in the complement regulatory proteins complement receptor 1 (CR1, CD35) and decay accelerating factor (DAF, CD55). We investigated whether these deficiencies affect the ability of erythrocytes to bind immune complexes (ICs) and regulate complement activation. We recruited 75 children with SMA (Hb < or = 6 g/dL) from the holoendemic malaria region of the Lake Victoria basin, western Kenya, and 74 age- and gender-matched uncomplicated malaria controls. In addition, we recruited 32 children with CM and 52 age- and gender-matched controls. Deficiencies in red cell CR1 and CD55 in children with SMA were accompanied by a marked decline in IC binding capacity and increased C3b deposition in vivo and ex vivo. Importantly, these changes were specific because they were not seen in red cells of children with CM or their controls. These data suggest that the declines in red cell CR1 and CD55 seen in children with SMA are of physiologic significance and may predispose erythrocytes to complement-mediated damage and phagocytosis in vivo.

Background: Severe anaemia (SA), intravascular haemolysis (IVH) and respiratory distress (RD) are severe forms of Plasmodium falciparum malaria, with RD reported to be of prognostic importance in African children with malarial anaemia. Complement factors have been implicated in the mechanism leading to excess anaemia in acute P. falciparum infection., Methods: The direct Coombs test (DCT) and flow cytometry were used to investigate the mean levels of RBC-bound complement fragments (C3d and C3balphabeta) and the regulatory proteins [complement receptor 1 (CD35) and decay accelerating factor (CD55)] in children with discrete clinical forms of P. falciparum malaria. The relationship between the findings and clinical parameters including coma, haemoglobin (Hb) levels and RD were investigated., Results: Of the 484 samples tested, 131(27%) were positive in DCT, out of which 115/131 (87.8%) were positive for C3d alone while 16/131 (12.2%) were positive for either IgG alone or both. 67.4% of the study population were below 5 years of age and DCT positivity was more common in this age group relative to children who were 5 years or older (Odds ratio, OR = 3.8; 95%CI, 2.2-6.7, p < 0.001). DCT correlated significantly with RD (beta = -304, p = 0.006), but multiple regression analysis revealed that, Hb (beta = -0.341, p = 0.012) and coma (beta = -0.256, p = 0.034) were stronger predictors of RD than DCT (beta = 0.228, p = 0.061). DCT was also not associated with IVH, p = 0.19, while spleen size was inversely correlated with Hb (r = -402, p = 0.001). Flow cytometry showed similar mean fluorescent intensity (MFI) values of CD35, CD55 and C3balphabeta levels on the surfaces of RBC in patients and asymptomatic controls (AC). However, binding of C3balphabeta correlated significantly with CD35 or CD55 (p < 0.001)., Conclusion: These results suggest that complement activation contributed to anaemia in acute childhood P. falciparum malaria, possibly through induction of erythrophagocytosis and haemolysis. In contrast to other studies, this study did not find association between levels of the complement regulatory proteins, CD35 and CD55 and malarial anaemia. These findings suggest that complement activation could also be involved in the pathogenesis of RD but larger studies are needed to confirm this finding.

Immune complexes (IC) are constantly formed at low levels in normal individuals. In humans, the red blood cell (RBC) complement receptor 1 (CR1) plays the dominant role in the IC binding and clearance. Over the last several years, we have investigated the potential utility of immunocamouflaged (methoxypoly(ethylene glycol) [mPEG] grafted) RBC to attenuate the risk of alloimmunization. Because the grafted polymer nonspecifically camouflages membrane proteins, its effects on CR1 detection and IC binding were assessed. The dose dependent (0-2.5 mM) effects of activated mPEG (CmPEG, 5 kDa; and BTCmPEG, 5 and 20 kDa) on CR1 detection and the binding of artificially generated IC [C3b coated alkaline phosphatase and antialkaline phosphatase complexes] to control and pegylated RBC was investigated by flow cytometry. In contrast to selected non-ABO blood group antigens, grafted mPEG did not effectively camouflage CR1. Surprisingly, however, even very low grafting concentrations of mPEG (>or=0.3 mM) resulted in a >or=95% loss in IC binding. Further reductions in grafting concentration (0.15 and 0.03 mM mPEG) still yielded decreased IC binding of approximately 60 and 40%, respectively. Importantly, unactivated mPEG had minimal effects on IC binding. These data demonstrate that even small amounts of grafted mPEG interfere with the multivalent CR1-IC interaction necessary for high affinity IC binding, hence large volume transfusions of mPEG-RBC may be contraindicated in patients with pre-existing IC disease. Whether this concern is of clinical significance in healthy humans is less clear due to dilutional effects and the presence of secondary clearance pathways., ((c) 2007 Wiley-Liss, Inc.)

The study of murine memory B cells has been limited by small cell numbers and the lack of a definitive marker. We have addressed some of these difficulties with hapten-specific transgenic (Tg) mouse models that yield relatively large numbers of antigen-specific memory B cells upon immunization. Using these models, along with a 5-bromo-2'-deoxyuridine (BrdU) pulse-label strategy, we compared memory cells to their naive precursors in a comprehensive flow cytometric survey, thus revealing several new murine memory B cell markers. Most interestingly, memory cells were phenotypically heterogeneous. Particularly surprising was the finding of an unmutated memory B cell subset identified by the expression of CD80 and CD35. We confirmed these findings in an analogous V region knock-in mouse and/or in non-Tg mice. There also was anatomic heterogeneity, with BrdU(+) memory cells residing not just in the marginal zone, as had been thought, but also in splenic follicles. These studies impact the current understanding of murine memory B cells by identifying new phenotypes and by challenging assumptions about the location and V region mutation status of memory cells. The apparent heterogeneity in the memory compartment implies either different origins and/or different functions, which we discuss.

Measurement of erythrocyte [red blood cells (RBC)] complement receptor type 1 (CR1, CD35) has the potential to serve as a sensitive assessment of complement activation and immune complex clearance. All previously reported monoclonal antibodies (MoAb) to the extracellular region of CR1 recognize epitopes within the long homologous repeats (LHR) of CR1 and the epitopes for the most frequently used MoAbs are repeated at least twice per CR1 molecule. Furthermore, CR1 exhibits structural polymorphism characterized by a variable number of LHR per molecule. Thus, accurate enumeration of cell surface CR1 using currently available MoAb would require that the results be corrected for the number of antibody epitopes per CR1 molecule encoded by each individual's alleles. To obtain a MoAb to a non-polymorphic epitope on human CR1, hybridomas were generated from mice immunized with recombinant soluble CR1 (sCR1) and MoAb were screened for those that recognized the full-length extracellular domain but failed to bind to all four recombinant LHR fragments. A single antibody, CR1-2B11, was identified and was found to recognize an epitope located wholly within SCR29-30 of CR1, NH2-terminal to an elastase cleavage site. Like other CR1 MoAb, the CR1-2B11 epitope expression decreased on old erythrocytes compared to younger cells and CR1-2B11 did not identify a CR1 'stump' on RBC. Importantly, CR1-2B11 immunofluorescence did not change with storage or handling of RBC, unlike the apparent decrease in immunofluorescence observed with other MoAb. CR1-2B11 should be useful for the accurate enumeration of RBC CR1.

B-1b cells produce IgM natural antibodies against alpha1-3Galbeta1-4GlcNAc (alphaGal). These can be tolerized by nonmyeloablative induction of mixed chimerism using alphaGal-positive (alphaGal+) donor marrow. We assessed the role of CR1/2 in this model for induction of tolerance of B-1b cells. Mixed hematopoietic chimerism was induced in alpha1-3galactosyltransferase (GalT-/-) and GalT-/-Cr2-/- mice with alphaGal+ BALB/c marrow donors. Anti-alphaGal Ab and anti-alphaGal Ab-producing B cells became undetectable in GalT-/- chimeras, whereas they persisted in chimeric GalT-/-Cr2-/- mice. To determine whether CR1/2 expression on stromal cells and/or hematopoietic cells was critical for B-1-cell tolerance, we generated GalT-/- radiation chimeras in which CR1/CR2 was expressed on either stromal cells, hematopoietic cells, neither, or both. After induction of mixed chimerism from alphaGal+ allogeneic bone marrow (BM) donors, anti-alphaGal-producing B cells were rendered tolerant in reconstituted recipients expressing only stromal CR1/CR2. Our results suggest a possible role for follicular dendritic cells that pick up immune complexes via CR1/CR2 receptors in the tolerization of B-1b cells.

Replication-defective herpes simplex virus (HSV) strains elicit durable immune responses and protect against virulent HSV challenge in mice, despite being unable to establish latent infection in neuronal cells. Mechanisms for generating long-lived immunity in the absence of viral persistence remain uncertain. In animals immunized with replication-defective HSV, durable serum immunoglobulin G (IgG) responses were elicited. Surprisingly, Western blot analyses revealed that the specificities of antiviral IgG changed over time, and antibody reactivity to some viral proteins was detected only very late. Thus, some of the durable IgG activity appeared to be contributed by either new or significantly enhanced antibody responses at late times. Following immunization, radiation bone marrow-chimeric mice lacking complement receptors CD21 and CD35 on stromal cells elicited only short-lived serum IgG and failed to mount recall responses to subsequent HSV exposure. Our results suggest that complement-mediated retention of viral antigens by stromal cells, such as follicular dendritic cells, is critical for optimal maintenance of antibody responses and B-cell memory following vaccination with replication-defective HSV.

Erythrocyte complement receptor type one (E-CR1) is thought to protect against immune complex (IC) disease through interactions that lead to E-CR1 consumption, and low E-CR1 levels are characteristic of systemic lupus erythematosus (SLE). The purpose of this study was to test the hypothesis that E-CR1 consumption can predict or mark SLE flare. Recurrently active SLE patients [n = 43; 28 with past or present major renal manifestations (SLER) and 15 without (SLENR)], were evaluated every 2 months by detailed protocol testing (mean follow-up 22 months), including direct measurements of E-CR1 levels using a radioimmunoassay. In all patients, detectable E-CR1 levels fluctuated widely through acute periods of consumption and regeneration, preventing the use of any single value as a baseline. However, when individual chronic baseline values were used, determined as the mean of all E-CR1 values 4 months or more from a flare, a clear trend was observed. In 16 of 16 instances of non-renal flare in SLER patients, E-CR1 levels decreased at flare (mean decrease 34%, P < 0.0001). In contrast, no consistent difference was observed for flare in SLENR patients or for renal flare in SLER patients. Changes in E-CR1 levels did not correlate with plasma CR1 levels. In conclusion, single occurrences of E-CR1 consumption did not generally predict or mark SLE flare. However, compared to the average E-CR1 levels measured during no-flare intervals, E-CR1 consumption in SLER patients at flare was strongly associated with freedom from signs of renal involvement. We postulate that E-CR1 consumption reflects E-CR1 function that includes protecting against SLE nephritis.

Complement receptors type 1 and 2 (CR1 (CD35)/CR2 (CD21)) are known to enhance the adaptive immune response. In mice, CR1/CR2 are expressed on B cells, follicular dendritic cells, and activated granulocytes. Recently, we showed that a subset of CD44high and CD62Llow T cells also expresses CR1 and CR2. We now report that CR1/CR2 are detectable on both CD4+ and CD8+ subsets of T cells. Lipopolysaccharide (LPS) from Gram-negative bacteria causes polyclonal activation of B cells and stimulation of macrophages and other antigen-presenting cells. We further demonstrate that LPS induced marked up-regulation of CD25 and CD69 on T cells from CR1/CR2 sufficient (Cr+/+), but significantly lower up-regulation on T cells from CR1/CR2 deficient (Cr-/-) mice. These findings point to a novel mechanism by which CR1/CR2 modulates the activation of T cells by LPS.

This longitudinal study investigates the change of erythrocyte complement receptor (E-CR1) expression in patients with severe acute respiratory syndrome (SARS). Circulating E-CR1 expression was semiquantified by flow cytometric analyses in 54 SARS patients and in 212 healthy individuals as a control. Since E-CR1 expression is influenced by the genetic polymorphisms in the CR1 gene, a major genetic polymorphism located within intron 27 of the CR1 gene was simultaneously analysed by polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP). The results showed that the expression level of E-CR1 (referred to as net fluorescence intensity values, NFI) was statistically correlated with the relevant genetic genotypes among the Chinese population including the healthy individuals (NFI: 5.14 +/- 0.82, 3.57 +/- 0.66 and 2.67 +/- 0.32 for HH, HL and LL genotypes, respectively) and SARS patients (NFI: 3.52 +/- 0.91 and 2.63 +/- 0.70 for HH and HL genotypes, respectively). Interestingly, the expression density of E-CR1 was found to fall significantly during the initiation and progressive phases (weeks 1 and 2 after the disease onset) and gradually returned close to normal through their whole convalescent phase (beginning from weeks 2 or 3 to weeks 7 or 8) in SARS patients irrespective CR1 genotype. In conclusion, our findings, at least, suggest that E-CR1 is likely involved in immune pathogenesis of SARS disease.

Defects of early complement components (C1, C4 and C2) are associated with the development of systemic lupus erythematosus (SLE). The availability of complement knockout mice has increased our knowledge on the role of complement in the regulation of adaptive immunity. An impaired removal of apoptotic bodies, a disturbed clearance of IgG immune complexes (ICs) and an insufficient B-cell regulation via complement receptors CD21/CD35 have been discussed as explanations for the induction of autoimmunity; however, a unifying hypothesis for the loss of B-cell tolerance in the absence of C1 or C4 is still lacking. Using IgM-containing ICs, we observed a significant accumulation of antigen within the splenic marginal zone (MZ) of C4-deficient mice but not in C3-deficient or complement receptors CD21/CD35-deficient mice. The targeting of antigen toward the MZ restored adaptive immunity (antibody response and class switch) in C4-deficient animals. A new explanation for the association of SLE and complement C4 deficiency would be that in the absence of C4, natural antibodies (IgM type) localize more self-antigen toward the MZ so that the auto-antibody response is increased and autoimmune disease ensues. As such, an inadequate localization of self-antigens might be responsible for the annulment of peripheral B-cell tolerance in the absence of C4.

Previous reports have identified a circulating pool of CD45(+) collagen I(+) CXCR4(+) (CD45(+)Col I(+)CXCR4(+)) cells, termed fibrocytes, that traffic to areas of fibrosis. No studies have demonstrated that these cells actually contribute to fibrosis, however. Pulmonary fibrosis was originally thought to be mediated solely by resident lung fibroblasts. Here we show that a population of human CD45(+)Col I(+)CXCR4(+) circulating fibrocytes migrates in response to CXCL12 and traffics to the lungs in a murine model of bleomycin-induced pulmonary fibrosis. Next, we demonstrated that murine CD45(+)Col I(+)CXCR4(+) fibrocytes also traffic to the lungs in response to a bleomycin challenge. Maximal intrapulmonary recruitment of CD45(+)Col I(+)CXCR4(+) fibrocytes directly correlated with increased collagen deposition in the lungs. Treatment of bleomycin-exposed animals with specific neutralizing anti-CXCL12 Ab's inhibited intrapulmonary recruitment of CD45(+)Col I(+)CXCR4(+) circulating fibrocytes and attenuated lung fibrosis. Thus, our results demonstrate, we believe for the first time, that circulating fibrocytes contribute to the pathogenesis of pulmonary fibrosis.

Erythrocytes bind immune complexes (ICs) composed of antibodies binding their respective antigen (e.g., bacteria, parasites, viruses, or autoantigen) plus complement proteins via complement receptors [immune adherence (IA)]. In vivo studies have shown that erythrocytes act as an inert shuttle, targeting ICs to fixed macrophages in liver and spleen. Here we outline established and emerging implications of IA in health and disease.

Complement-mediated opsonization of encapsulated Staphylococcus aureus (CP+) of the predominant capsule types, 5 and 8, remains poorly understood. Our previous work showed that complement is important for mouse survival of CP+ type 5 bacteremia and that the type 5 capsule inhibits the binding of opsonic C3 fragments to the organism. The importance of complement-mediated opsonization of CP+ was tested by neutrophil phagocytosis assays. Complement-mediated opsonization of CP+ increased phagocytosis by 57% compared to opsonization in complement-inhibited serum. Agar-grown CP+, enhancing capsule expression, was phagocytosed only one-tenth as well as the capsule-negative organisms (CP-), supporting the belief that staphylococcal polysaccharide capsules impair phagocytosis. Despite relatively poor phagocytosis of CP+ compared to CP-, complement activation increased the phagocytosis of CP+ by 103%. Thus, complement in normal human serum may have an important role in opsonizing CP+, even when capsule expression is strong. The ability of bound C3 fragments to interact with complement receptor 1 (CD35) on the membrane of human erythrocytes was tested in an immune adherence assay. S. aureus capsule was able to mask C3 fragments on the organism from binding to complement receptor 1. The inhibition of C3 binding to CP+ and the masking of deposited C3 fragments caused by the presence of capsule was associated with markedly decreased phagocytosis. The addition of anti-capsule antibodies to normal human serum was found to markedly improve the recognition of deposited C3 fragments by complement receptor 1 even when the absolute number of C3 molecules bound to S. aureus was not increased.

The acquired loss of CR1 (CD35) on erythrocytes in specific autoimmune diseases and chronic infections may be due to autoAb against CR1. An ELISA using rCR1 was established to measure antiCR1 IgG autoAb. Plasma containing alloAb to polymorphism on CR1 (Knops blood group Ab) reacted strongly against rCR1 and were used as positive controls. AntiCR1 Ab was found in 3/90 (3.5%) plasma samples from healthy blood donors. The binding of these Ab was not inhibited by high salt concentrations. AntiCR1 Ab were present in the IgG fractions of plasma, and they bound to rCR1 on Western Blot. Affinity chromatography on rCR1-sepharose depleted the plasma of antiCR1, and the acid-eluted fractions contained the antiCR1 Ab. An increased frequency of antiCR1 autoAb was found in patients with SLE (36/78; 46%), liver cirrhosis (15/41; 36%), HIV infection (23/76; 30%) (all P < 0.0001), and in patients with anticardiolipin Ab (4/21; 19%, P < 0.01) multiple sclerosis (7/50; 14%, P < 0.02), and myeloma (autoAb (8/56; 14%, P < 0.02), but not in those with acute poststreptococcal glomerulonephritis (1:32; 3%). Because C1q binds to CR1, antiC1q Ab were analysed in the same patients. There was no correlation between levels of antiC1q and antiCR1 autoAb. In HIV patients, levels of antiCR1 did not correlate with low CR1 levels expressed on erythrocytes or soluble CR1 in plasma. The binding of antiCR1 autoAb to rCR1 fixed on ELISA plates was not inhibited by soluble rCR1 or by human erythrocyte CR1, in contrast to alloAb and one SLE serum, which induced partial blockade. Thus, antiCR1 autoAb recognize mostly CR1 epitope(s) not present on the native molecule, suggesting that they are not directly involved in the loss of CR1. Rather antiCR1 autoAb might indicate a specific immune response to denatured CR1.

The CD21/35 receptor provides an important link between innate and adaptive immunity. Its importance during protective immune responses to encapsulated extracellular bacteria was assessed using a new line of mice completely deficient in CD21/35 expression (CD21/35(-/-)). CD21/35 expression was essential for the rapid trapping of C3dg-antigen complexes by B cells in vivo, especially in splenic marginal zones. Despite normal B cell development in CD21/35(-/-) mice, T cell-independent and -dependent antibody responses to low-dose antigens were significantly decreased, with a striking impairment in IgG3 responses. Accordingly, CD21/35(-/-) mice were more susceptible to acute lethal Streptococcus pneumoniae infection. Thus, CD21/35 expression is critical for early protective antibody responses to lethal pathogens that rapidly multiply and quickly overwhelm the immune system.

To dissect the influence of CD21/CD35 and FcgammaRIIB in antigen retention and humoral memory, we used an adoptive transfer model in which antigen-primed B and T lymphocytes were given to sublethally irradiated wild-type mice or mice deficient in CD21/CD35 (Cr2(-/-)) or FcgammaRIIB receptors (FcgammaRIIB(-/-)). Cr2(-/-) chimeras showed impaired memory as characterized by a decrease in antibody titer, reduced frequency of antibody secreting cells, an absence of affinity maturation, and significantly reduced recall response. The impaired memory in Cr2(-/-) chimeras corresponded with the reduced frequency of antigen-specific memory B cells. Interestingly, FcgammaRIIB(-/-) chimeras showed a differential phenotype with impaired splenic but normal bone marrow responses. These data suggest that CD21/CD35 on stroma, including follicular dendritic cells, is critical to the maintenance of long-term B lymphocyte memory.

The cecropin-like bactericidal peptide Hp(2-20) from Helicobacter pylori induces activation of the NADPH oxidase in human neutrophils via formyl peptide receptor-like 1 (FPRL1) (J. Bylund, T. Christophe, F. Boulay, T. Nyström, A. Karlsson, and C. Dahlgren, Antimicrob. Agents Chemother. 45:1700-1704, 2001). Here we investigated the ability of bacterial lipopolysaccharide (LPS) to prime this response. Neutrophils treated with LPS for 30 min at 37 degrees C produced substantially more superoxide anion than control cells upon stimulation with Hp(2-20). Hence, LPS primed the cells for subsequent stimulation through FPRL1. To study the molecular background of this priming phenomenon, we measured the degrees of granule mobilization and concomitant receptor upregulation to the cell surface in LPS-treated cells. Exposure of complement receptors 1 and 3 as well as the formyl peptide receptor (FPR) was markedly increased after LPS treatment. Since approximately 60% of the gelatinase granules were mobilized while the specific granules were retained, we hypothesized that the gelatinase granules were potential stores of FPRL1. The presence of FPRL1 mainly in the gelatinase granules was confirmed by Western blotting of subcellular fractions of resting neutrophils. These results suggest that the mechanism behind the LPS-induced priming of FPRL1-mediated responses lies at the level of granule (receptor) mobilization.

Autoreactive B cells are present in the lymphoid tissues of healthy individuals, but typically remain quiescent. When this homeostasis is perturbed, the formation of self-reactive antibodies can have serious pathological consequences. B cells expressing an antigen receptor specific for self-immunoglobulin-gamma (IgG) make a class of autoantibodies known as rheumatoid factor (RF). Here we show that effective activation of RF+ B cells is mediated by IgG2a-chromatin immune complexes and requires the synergistic engagement of the antigen receptor and a member of the MyD88-dependent Toll-like receptor (TLR) family. Inhibitor studies implicate TLR9. These data establish a critical link between the innate and adaptive immune systems in the development of systemic autoimmune disease and explain the preponderance of autoantibodies reactive with nucleic acid-protein particles. The unique features of this dual-engagement pathway should facilitate the development of therapies that specifically target autoreactive B cells.

Normal human B lymphocytes activate the alternative pathway of complement via complement receptor type 2 (CR2, CD21), that binds hydrolysed C3 (iC3) and thereby promotes the formation of a membrane-bound C3 convertase. We have investigated whether this might lead to the generation of a C5 convertase and consequent formation of membrane attack complexes (MAC). Deposition of C3 fragments and MAC was assessed on human peripheral B lymphocytes in the presence of 30% autologous serum containing 4.4 mM MgCl2/20 mM EGTA, which abrogates the classical pathway of complement without affecting the alternative pathway. Blockade of the CR2 ligand-binding site with the monoclonal antibody FE8 resulted in 56 +/- 13% and 71 +/- 9% inhibition of the C3-fragment and MAC deposition, respectively, whereas the monoclonal antibody HB135, directed against an irrelevant CR2 epitope, had no effect. Blockade of the CR1 binding site with the monoclonal antibody 3D9 also resulted in a minor reduction in MAC deposition, while FE8 and 3D9, in combination, markedly reduced deposition of both C3 fragments (91 +/- 5%) and C9 (95 +/- 3%). The kinetics of C3-fragment and MAC deposition, as well as the dependence of both processes on CR2, indicate that MAC formation is a consequence of alternative pathway activation.