Complement activation drives antibody-mediated transfusion-related acute lung injury via macrophage trafficking and formation of NETs.

ABSTRACT: Transfusion-related acute lung injury (TRALI) is one of the leading causes of transfusion-related fatalities and, to date, is without available therapies. Here, we investigated the role of the complement system in TRALI. Murine anti-major histocompatibility complex class I antibodies were used in TRALI mouse models, in combination with analyses of plasma samples from patients with TRALI. We found that in vitro complement activation was related to in vivo antibody-mediated TRALI induction, which was correlated with increased macrophage trafficking from the lungs to the blood in a fragment crystallizable region (Fc)-dependent manner and that this was dependent on C5. Human immunoglobulin G 1 variants of the murine TRALI-inducing antibody 34-1-2S, either unable to activate complement and/or bind to Fcγ receptors (FcγRs), revealed an essential role for the complement system, but not for FcγRs, in the onset of 34-1-2S-mediated TRALI in mice. In addition, we found high levels of complement activation in the plasma of patients with TRALI (n = 53), which correlated with elevated neutrophil extracellular trap (NET) markers. In vitro we found that NETs could be formed in a murine, 2-hit model, mimicking TRALI with lipopolysaccharide and C5a stimulation. Collectively, this reveals a critical role of Fc-mediated complement activation in TRALI, with a direct relation to macrophage trafficking from the lungs to the blood and an association with NET formation, suggesting that targeting the complement system may be an attractive therapeutic approach for combating TRALI.

Variation in CFHR3 determines susceptibility to meningococcal disease by controlling factor H concentrations.

Neisseria meningitidis protects itself from complement-mediated killing by binding complement factor H (FH). Previous studies associated susceptibility to meningococcal disease (MD) with variation in CFH, but the causal variants and underlying mechanism remained unknown. Here we attempted to define the association more accurately by sequencing the CFH-CFHR locus and imputing missing genotypes in previously obtained GWAS datasets of MD-affected individuals of European ancestry and matched controls. We identified a CFHR3 SNP that provides protection from MD (rs75703017, p value = 1.1 × 10-16) by decreasing the concentration of FH in the blood (p value = 1.4 × 10-11). We subsequently used dual-luciferase studies and CRISPR gene editing to establish that deletion of rs75703017 increased FH expression in hepatocyte by preventing promotor inhibition. Our data suggest that reduced concentrations of FH in the blood confer protection from MD; with reduced access to FH, N. meningitidis is less able to shield itself from complement-mediated killing.

Unraveling the Effect of a Potentiating Anti-Factor H Antibody on Atypical Hemolytic Uremic Syndrome-Associated Factor H Variants.

The complement system plays an important role in our innate immune system. Complement activation results in clearance of pathogens, immune complex, and apoptotic cells. The host is protected from complement-mediated damage by several complement regulators. Factor H (FH) is the most important fluid-phase regulator of the alternative pathway of the complement system. Heterozygous mutations in FH are associated with complement-related diseases such as atypical hemolytic uremic syndrome (aHUS) and age-related macular degeneration. We recently described an agonistic anti-FH mAb that can potentiate the regulatory function of FH. This Ab could serve as a potential new drug for aHUS patients and alternative to C5 blockade by eculizumab. However, it is unclear whether this Ab can potentiate FH mutant variants in addition to wild-type (WT) FH. In this study, the functionality and potential of the agonistic Ab in the context of pathogenic aHUS-related FH mutant proteins was investigated. The binding affinity of recombinant WT FH and the FH variants, W1183L, V1197A, R1210C, and G1194D to C3b was increased upon addition of the potentiating Ab and similarly, the decay-accelerating activity of all mutants is increased. The potentiating anti-FH Ab is able to restore the surface regulatory function of most of the tested FH mutants to WT FH levels on a human HAP-1 cell line and on sheep erythrocytes. In conclusion, our potentiating anti-FH is broadly active and able to enhance both WT FH function as well as most aHUS-associated FH variants tested in this study.

Complement activation by salivary agglutinin is secretor status dependent.

After mucosal damage or gingival inflammation, complement proteins leak into the oral cavity and mix with salivary proteins such as salivary agglutinin (SAG/gp-340/DMBT1). This protein is encoded by the gene Deleted in Malignant Brain Tumors 1 (DMBT1), and it aggregates bacteria, viruses and fungi, and activates the lectin pathway of the complement system. In the lectin pathway, carbohydrate structures on pathogens or altered self cells are recognized. SAG is highly glycosylated, partly on the basis of the donor's blood group status. Whereas secretors express Lewis b, Lewis y, and antigens from the ABO-blood group system on SAG, non-secretors do not. Through mannose-binding lectin (MBL) binding and C4 deposition assays, we aimed to identify the chemical structures on SAG that are responsible for complement activation. The complement-activating properties of SAG were completely abolished by oxidation of its carbohydrate moiety. SAG-mediated activation of complement was also inhibited in the presence of saccharides such as fucose and Lewis b carbohydrates, and also after pretreatment with the fucose-binding lectin, Anguilla anguilla agglutinin. Complement activation was significantly (p<0.01) higher in secretors than in non-secretors. Our results suggest that fucose-rich oligosaccharide sidechains, such as Lewis b antigens, are involved in the activation of complement by SAG.

Platelets interact with CD169+ macrophages and cDC1 and enhance liposome-induced CD8+ T cell responses.

Historically platelets are mostly known for their crucial contribution to hemostasis, but there is growing understanding of their role in inflammation and immunity. The immunomodulatory role of platelets entails interaction with pathogens, but also with immune cells including macrophages and dendritic cells (DCs), to activate adaptive immune responses. In our previous work, we have demonstrated that splenic CD169+ macrophages scavenge liposomes and collaborate with conventional type 1 DCs (cDC1) to induce expansion of CD8+ T cells. Here, we show that platelets associate with liposomes and bind to DNGR-1/Clec9a and CD169/Siglec-1 receptors in vitro. In addition, platelets interacted with splenic CD169+ macrophages and cDC1 and further increased liposome internalization by cDC1. Most importantly, platelet depletion prior to liposomal immunization resulted in significantly diminished antigen-specific CD8+ T cell responses, but not germinal center B cell responses. Previously, complement C3 was shown to be essential for platelet-mediated CD8+ T cell activation during bacterial infection. However, after liposomal vaccination CD8+ T cell priming was not dependent on complement C3. While DCs from platelet-deficient mice exhibited unaltered maturation status, they did express lower levels of CCR7. In addition, in the absence of platelets, CCL5 plasma levels were significantly reduced. Overall, our findings demonstrate that platelets engage in a cross-talk with CD169+ macrophages and cDC1 and emphasize the importance of platelets in induction of CD8+ T cell responses in the context of liposomal vaccination.

The contribution of the alternative pathway in complement activation on cell surfaces depends on the strength of classical pathway initiation.

Objectives: The complement system is an important component of innate immunity. The alternative pathway (AP) amplification loop is considered an essential feed forward mechanism for complement activation. However, the role of the AP in classical pathway (CP) activation has only been studied in ELISA settings. Here, we investigated its contribution on physiologically relevant surfaces of human cells and bacterial pathogens and in antibody-mediated complement activation, including in autoimmune haemolytic anaemia (AIHA) setting with autoantibodies against red blood cells (RBCs). Methods: We evaluated the contribution of the AP to complement responses initiated through the CP on human RBCs by serum of AIHA patients and recombinant antibodies. Moreover, we studied complement activation on Neisseria meningitidis and Escherichia coli. The effect of the AP was examined using either AP-depleted sera or antibodies against factor B and factor D. Results: We show that the amplification loop is redundant when efficient CP activation takes place. This is independent of the presence of membrane-bound complement regulators. The role of the AP may become significant when insufficient CP complement activation occurs, but this depends on antibody levels and (sub)class. Our data indicate that therapeutic intervention in the amplification loop will most likely not be effective to treat antibody-mediated diseases. Conclusion: The AP can be bypassed through efficient CP activation. The AP amplification loop has a role in complement activation during conditions of modest activation via the CP, when it can allow for efficient complement-mediated killing.

Low Levels of Factor H Family Proteins During Meningococcal Disease Indicate Systemic Processes Rather Than Specific Depletion by Neisseria meningitidis.

Neisseria meningitidis, the causative agent of meningococcal disease (MD), evades complement-mediated clearance upon infection by 'hijacking' the human complement regulator factor H (FH). The FH protein family also comprises the homologous FH-related (FHR) proteins, hypothesized to act as antagonists of FH, and FHR-3 has recently been implicated to play a major role in MD susceptibility. Here, we show that the circulating levels of all FH family proteins, not only FH and FHR-3, are equally decreased during the acute illness. We did neither observe specific consumption of FH or FHR-3 by N. meningitidis, nor of any of the other FH family proteins, suggesting that the globally reduced levels are due to systemic processes including dilution by fluid administration upon admission and vascular leakage. MD severity associated predominantly with a loss of FH rather than FHRs. Additionally, low FH levels associated with renal failure, suggesting insufficient protection of host tissue by the active protection by the FH protein family, which is reminiscent of reduced FH activity in hemolytic uremic syndrome. Retaining higher levels of FH may thus limit tissue injury during MD.

Mechanisms Driving Neutrophil-Induced T-cell Immunoparalysis in Ovarian Cancer.

T-cell activation and expansion in the tumor microenvironment (TME) are critical for antitumor immunity. Neutrophils in the TME acquire a complement-dependent T-cell suppressor phenotype that is characterized by inhibition of T-cell proliferation and activation through mechanisms distinct from those of myeloid-derived suppressor cells. In this study, we used ascites fluid supernatants (ASC) from patients with ovarian cancer as an authentic component of the TME to evaluate the effects of ASC on neutrophil function and mechanisms for neutrophil-driven immune suppression. ASC prolonged neutrophil life span, decreased neutrophil density, and induced nuclear hypersegmentation. Mass cytometry analysis showed that ASC induced 15 distinct neutrophil clusters. ASC stimulated complement deposition and signaling in neutrophils, resulting in surface mobilization of granule constituents, including NADPH oxidase. NADPH oxidase activation and phosphatidylserine signaling were required for neutrophil suppressor function, although we did not observe a direct role of extracellular reactive oxygen species in inhibiting T-cell proliferation. Postoperative surgical drainage fluid also induced a complement-dependent neutrophil suppressor phenotype, pointing to this effect as a general response to injury. Like circulating lymphocytes, ASC-activated neutrophils caused complement-dependent suppression of tumor-associated lymphocytes. ASC-activated neutrophils adhered to T cells and caused trogocytosis of T-cell membranes. These injury and signaling cues resulted in T-cell immunoparalysis characterized by impaired NFAT translocation, IL2 production, glucose uptake, mitochondrial function, and mTOR activation. Our results demonstrate that complement-dependent priming of neutrophil effector functions in the TME induces a T-cell nonresponsiveness distinct from established checkpoint pathways and identify targets for immunotherapy.See related Spotlight by Cassatella, p. 725.

Studies on the haemolytic activity of circulating C1q-C3/C4 complexes.

During classical complement pathway activation, the internal thio-ester of both C3 and C4 becomes exposed which enables C3 and C4 to bind covalently to nearby molecules. Recently, we described that C3 and C4 bind to C1q, the recognition molecule of the classical pathway, upon activation of this pathway. Covalently linked complexes between C1q and activated C4 (C1q-C4 complexes) are specific markers for classical complement pathway activation. In the present study we further investigated the molecular characteristics of complexes between C1q and activated C3 or C4 that occur in vivo. In human serum only complexes of C1q with C3d or C4d fragments were detected but not those with the larger C3b/bi or C4b/bi fragments. We identified that C1q-C4 complexes circulate as part of the intact C1 complex instead of as free C1q. Finally, we investigated whether deposited C3d or C4d affect C1 haemolytic activity. We observed that both C1q-C3 and C1q-C4 complexes are significantly (P<0.05) less active in a C1q-haemolytic assay than non-complexed C1q. Thus, the dominant types of C1q complexes that circulate in vivo are C1q-C3d and C1q-C4d complexes. These complexes are still able to interact with C1r and C1s to form a C1 complex, but seem to have a reduced activity as compared to C1q not carrying C3- or C4-fragments.

Potentiation of complement regulator factor H protects human endothelial cells from complement attack in aHUS sera.

Mutations in the gene encoding for complement regulator factor H (FH) severely disrupt its normal function to protect human cells from unwanted complement activation, resulting in diseases such as atypical hemolytic uremic syndrome (aHUS). aHUS presents with severe hemolytic anemia, thrombocytopenia, and renal disease, leading to end-stage renal failure. Treatment of severe complement-mediated disease, such as aHUS, by inhibiting the terminal complement pathway, has proven to be successful but at the same time fails to preserve the protective role of complement against pathogens. To improve complement regulation on human cells without interfering with antimicrobial activity, we identified an anti-FH monoclonal antibody (mAb) that induced increased FH-mediated protection of primary human endothelial cells from complement, while preserving the complement-mediated killing of bacteria. Moreover, this FH-activating mAb restored complement regulation in sera from aHUS patients carrying various heterozygous mutations in FH known to impair FH function and dysregulate complement activation. Our data suggest that FH normally circulates in a less active conformation and can become more active, allowing enhanced complement regulation on human cells. Antibody-mediated potentiation of FH may serve as a highly effective approach to inhibit unwanted complement activation on human cells in a wide range of hematological diseases while preserving the protective role of complement against pathogens.

Complement Factor H-Related Protein 4A Is the Dominant Circulating Splice Variant of CFHR4.

Recent research has elucidated circulating levels of almost all factor H-related (FHR) proteins. Some of these proteins are hypothesized to act as antagonists of the important complement regulator factor H (FH), fine-tuning complement regulation on human surfaces. For the CFHR4 splice variants FHR-4A and FHR-4B, the individual circulating levels are unknown, with only total levels being described. Specific reagents for FHR-4A or FHR-4B are lacking due to the fact that the unique domains in FHR-4A show high sequence similarity with FHR-4B, making it challenging to distinguish them. We developed an assay that specifically measures FHR-4A using novel, well-characterized monoclonal antibodies (mAbs) that target unique domains in FHR-4A only. Using various FHR-4A/FHR-4B-specific mAbs, no FHR-4B was identified in any of the serum samples tested. The results demonstrate that FHR-4A is the dominant splice variant of CFHR4 in the circulation, while casting doubt on the presence of FHR-4B. FHR-4A levels (avg. 2.55 ± 1.46 µg/mL) were within the range of most of the previously reported levels for all other FHRs. FHR-4A was found to be highly variable among the population, suggesting a strong genetic regulation. These results shed light on the physiological relevance of the previously proposed role of FHR-4A and FHR-4B as antagonists of FH in the circulation.

Complement Factor H Levels Associate With Plasmodium falciparum Malaria Susceptibility and Severity.

BACKGROUND: Plasmodium falciparum may evade complement-mediated host defense by hijacking complement Factor H (FH), a negative regulator of the alternative complement pathway. Plasma levels of FH vary between individuals and may therefore influence malaria susceptibility and severity. METHODS: We measured convalescent FH plasma levels in 149 Gambian children who had recovered from uncomplicated or severe P. falciparum malaria and in 173 healthy control children. We compared FH plasma levels between children with malaria and healthy controls, and between children with severe (n = 82) and uncomplicated malaria (n = 67). We determined associations between FH plasma levels and laboratory features of severity and used multivariate analyses to examine associations with FH when accounting for other determinants of severity. RESULTS: FH plasma levels differed significantly between controls, uncomplicated malaria cases, and severe malaria cases (mean [95% confidence interval], 257 [250 to 264], 288 [268 to 309], and 328 [313 to 344] µg/mL, respectively; analysis of variance P < .0001). FH plasma levels correlated with severity biomarkers, including lactate, parasitemia, and parasite density, but did not correlate with levels of PfHRP2, which represent the total body parasite load. Associations with severity and lactate remained significant when adjusting for age and parasite load. CONCLUSIONS: Natural variation in FH plasma levels is associated with malaria susceptibility and severity. A prospective study will be needed to strengthen evidence for causation, but our findings suggest that interfering with FH binding by P. falciparum might be useful for malaria prevention or treatment.

The development of rat Leydig cell progenitors in vitro: how essential is luteinising hormone?

UNLABELLED: Luteinising hormone (LH) appears to be important for the establishment of the adult-type Leydig cell population. The role of LH in the initial steps of stem Leydig cell/precursor cell differentiation is less clear. The aim of the present study was to elucidate the role of LH in the differentiation of spindle-shaped mesenchymal-like cells into Leydig cell progenitors. Interstitial cells were isolated from rat testes at three different ages reflecting different phases in development, and cultured in the presence of increasing concentrations of LH (ranging from 0.01 to 10 ng/ml). Cells were isolated from 10-day-old rats when stem Leydig cells/precursor cells are abundant; 13-day-old rats when the first 3beta-hydroxysteroid dehydrogenase (3beta-HSD)-positive Leydig cell progenitors have developed in the strain of rats used in this study; and 18-day-old rats just prior to the wave of progenitor proliferation. Immunohistochemistry revealed that before stem Leydig cells differentiate into progenitor cells, they acquire functional LH receptors and become precursor cells. This was confirmed by an in vivo immunohistochemical double-labelling experiment. Addition of LH to the cultures increased the percentage of LH/3beta-HSD-labelled Leydig cell progenitors, while the percentage of cells solely expressing the LH receptor decreased. Cell proliferation was negligible, suggesting that the increase in 3beta-HSD-positive cells is the result of precursor cell differentiation. When interstitial cells were isolated from 13-day-old rats and to a lesser extent from 10-day-old rats, a small proportion of the precursors could develop into progenitor cells independent of the presence of LH. IN CONCLUSION: before Leydig stem cells differentiate into 3beta-HSD-positive progenitor cells, they acquire LH receptors and become precursor cells. LH appears to be essential, even at very low doses for the differentiation of these precursor cells into 3beta-HSD-positive progenitors, although a subpopulation of precursor cells can develop into progenitors independently of LH.

Oncostatin-M inhibits luteinizing hormone stimulated Leydig cell progenitor formation in vitro.

BACKGROUND: The initial steps of stem Leydig cell differentiation into steroid producing progenitor cells are thought to take place independent of luteinizing hormone (LH), under the influence of locally produced factors such as leukaemia inhibitory factor (LIF), platelet derived growth factor A and stem cell factor. For the formation of a normal sized Leydig cell population in the adult testis, the presence of LH appears to be essential. Oncostatin M (OSM) is a multifunctional cytokine and member of the interleukin (IL)-6 family that also includes other cytokines such as LIF. In the rat OSM is highly expressed in the late fetal and neonatal testis, and may thus be a candidate factor involved in Leydig cell progenitor formation. METHODS: Interstitial cells were isolated from 13-day-old rat testes and cultured for 1, 3 or 8 days in the presence of different doses of OSM (range: 0.01 to 10 ng/ml) alone or in combination with LH (1 ng/ml). The effects of OSM and LH on cell proliferation were determined by incubating the cultures with [3H]thymidine or bromodeoxyuridine (BrdU). Developing progenitor cells were identified histochemically by the presence of the marker enzyme 3beta-hydroxysteroid dehydrogenase (3beta-HSD). RESULTS: OSM, when added at a dose of 10 ng/ml, caused a nearly 2-fold increase in the percentage of Leydig cell progenitors after 8 days of culture. Immunohistochemical double labelling experiments with 3beta-HSD and BrdU antibodies showed that this increase was the result of differentiation of stem Leydig cells/precursor cells and not caused by proliferation of progenitor cells themselves. The addition of LH to the cultures consistently resulted in an increase in progenitor formation throughout the culture period. Surprisingly, when OSM and LH were added together, the LH induced rise in progenitor cells was significantly inhibited after 3 and 8 days of culture. CONCLUSION: Taken together, the results of the present study suggest that locally produced OSM may not only play a role in the regulation of Sertoli cell proliferation and the initiation of spermatogenesis but may also play a role in the regulation of Leydig cell progenitor formation by keeping the augmenting effects of LH on this process in abeyance.

Factor H-Related (FHR)-1 and FHR-2 Form Homo- and Heterodimers, while FHR-5 Circulates Only As Homodimer in Human Plasma.

The complement factor H-related (FHR) proteins are hypothesized to fine-tune the regulatory role of complement factor H (FH) in the alternative pathway of the complement system. Moreover, FHR-1, FHR-2, and FHR-5 have been proposed to be dimers, which further complicates accurate analysis. As FHRs are highly similar among themselves and toward FH, obtaining specific reagents for quantification of serum levels and functional analysis is challenging. In this study, we generated antibodies and developed ELISAs to measure FHR-1, FHR-2, and FHR-5 in serum. We used both recombinant and serum-derived proteins to show that four dimers occur in human circulation: homodimers of FHR-1, FHR-2, and FHR-5, as well as FHR-1/FHR-2 heterodimers. Heterodimers containing FHR-5 were not found. In individuals with homozygous CFHR1 deletions or compound heterozygous CFHR2 missense/nonsense mutations identified in this study, the respective FHR-1 and FHR-2 homo- and heterodimers were absent. Using FRET, we found that recombinant FHR dimers exchange monomers rapidly. This was confirmed ex vivo, using FHR-1- and FHR-2-deficient sera. Of all FHR dimers, FHR-5/5 homodimers demonstrated strong binding affinity toward heparin. Specific ELISAs demonstrated that serum levels of FHR-1/1, FHR-1/2, FHR-2/2, and FHR-5/5 dimers were low compared to FH, which circulates at a 10- to 200-fold molar excess. In summary, FHR-1, FHR-2, and FHR-5 homodimerize, with FHR-1 and FHR-2 forming heterodimers as well, and equilibrate quickly in plasma.

Decoding the Human Immunoglobulin G-Glycan Repertoire Reveals a Spectrum of Fc-Receptor- and Complement-Mediated-Effector Activities.

Glycosylation of the immunoglobulin G (IgG)-Fc tail is required for binding to Fc-gamma receptors (FcγRs) and complement-component C1q. A variety of IgG1-glycoforms is detected in human sera. Several groups have found global or antigen-specific skewing of IgG glycosylation, for example in autoimmune diseases, viral infections, and alloimmune reactions. The IgG glycoprofiles seem to correlate with disease outcome. Additionally, IgG-glycan composition contributes significantly to Ig-based therapies, as for example IVIg in autoimmune diseases and therapeutic antibodies for cancer treatment. The effect of the different glycan modifications, especially of fucosylation, has been studied before. However, the contribution of the 20 individual IgG glycoforms, in which the combined effect of all 4 modifications, to the IgG function has never been investigated. Here, we combined six glyco-engineering methods to generate all 20 major human IgG1-glycoforms and screened their functional capacity for FcγR and complement activity. Bisection had no effect on FcγR or C1q-binding, and sialylation had no- or little effect on FcγR binding. We confirmed that hypo-fucosylation of IgG1 increased binding to FcγRIIIa and FcγRIIIb by ~17-fold, but in addition we showed that this effect could be further increased to ~40-fold for FcγRIIIa upon simultaneous hypo-fucosylation and hyper-galactosylation, resulting in enhanced NK cell-mediated antibody-dependent cellular cytotoxicity. Moreover, elevated galactosylation and sialylation significantly increased (independent of fucosylation) C1q-binding, downstream complement deposition, and cytotoxicity. In conclusion, fucosylation and galactosylation are primary mediators of functional changes in IgG for FcγR- and complement-mediated effector functions, respectively, with galactose having an auxiliary role for FcγRIII-mediated functions. This knowledge could be used not only for glycan profiling of clinically important (antigen-specific) IgG but also to optimize therapeutic antibody applications.

Estrous cycle dependent changes in expression and distribution of Fas, Fas ligand, Bcl-2, Bax, and pro- and active caspase-3 in the rat ovary.

In the present investigation, the localization of proteins involved in ovarian apoptosis were studied throughout the estrous cycle in the presence of fluctuating hormone levels. Fas, Fas ligand, Bcl-2, Bax and caspase-3 mRNA expression and proteins were detected in all ovarian tissue extracts, though the amount of protein varied with the phase of the estrous cycle. Fas, Bax and caspase-3 protein levels were highest at diestrus and decreased thereafter towards metestrus. In contrast, Fas ligand and Bcl-2 protein levels were lowest at diestrus and increased toward metestrus. Immunohistochemistry revealed that the staining of the anti-apoptotic protein Bcl-2 was more pronounced in healthy preantral follicles than in atretic follicles. In contrast, the pro-apoptotic proteins Fas, Fas ligand, Bax and active caspase-3 were more predominantly present in atretic follicles. In the ovarian surface epithelium (OSE), Fas, procaspase-3 and Bcl-2 immunostaining appeared independent of the phase of the estrous cycle. Fas ligand and Bax staining was detected particularly during proestrus in OSE cells surrounding the ovulatory follicles, while active caspase-3 was observed only in OSE cells at the postovulatory site during estrus. The proportion of luteal cells that stained positively for Fas, Bax and caspase-3 increased with the age of the corpus luteum, while Fas ligand and Bcl-2 immunostaining was strongest in newly formed corpora lutea and decreased thereafter. In conclusion, the components of the Fas signalling pathway were differentially expressed throughout the estrous cycle in a variety of ovarian cell types, which may correspond to hormone dependent survival mechanisms.

Salivary agglutinin is the major component in human saliva that modulates the lectin pathway of the complement system.

Saliva interacts with blood after mucosal damage or leakage of gingival crevicular fluid. Surface-adsorbed salivary agglutinin (SAG) activates the lectin pathway (LP) of the complement system via mannose-binding lectin, while SAG in solution inhibits complement activation. In the present study we investigated if, next to SAG, whole and glandular saliva itself and other salivary glycoproteins activate or inhibit the LP. Complement activation was measured by detecting C4 deposition on microtiter plates coated with saliva or purified proteins. Complement inhibition was measured after incubating serum with saliva or proteins in microtiter plates coated with mannan, an LP activator. Adsorbed whole, sublingual and submandibular saliva showed LP-dependent complement activation. Blood group secretors, but not non-secretors, activated the LP. Saliva of both secretors and non-secretors inhibited C4 deposition on mannan. After depletion of SAG, saliva no longer inhibited the LP. Other salivary proteins, including amylase, MUC5B and histatin 2, did not activate or inhibit the LP. Surface-adsorbed whole saliva and glandular saliva samples activate the LP of complement, depending on the presence of SAG and the secretor status of the donor. In solution, saliva inhibits the LP, depending on the presence of SAG, but independent of the secretor status.

Complement Factor H-Related Protein 3 Serum Levels Are Low Compared to Factor H and Mainly Determined by Gene Copy Number Variation in CFHR3.

The major human complement regulator in blood, complement factor H (FH), has several closely related proteins, called FH-related (FHR) proteins. As all FHRs lack relevant complement regulatory activity, their physiological role is not well understood. FHR protein 3 (FHR-3) has been suggested to compete with FH for binding to Neisseria meningitidis, thereby affecting complement-mediated clearance. Clearly, the in vivo outcome of such competition greatly depends on the FH and FHR-3 concentrations. While FH levels have been established, accurate FHR-3 levels were never unequivocally reported to date. Moreover, CFHR3 gene copy numbers commonly vary, which may impact the FHR-3 concentration. Hence, we generated five anti-FHR-3 mAbs to specifically measure FHR-3 in human healthy donors of which we determined the gene copy number variation at the CFH/CFHR locus. Finally, we examined the acute-phase response characteristics of FHR-3 in a small sepsis cohort. We determined FHR-3 levels to have a mean of 19 nM and that under normal conditions the copy number of CFHR3 correlates to a very large extent with the FHR-3 serum levels. On average, FHR-3 was 132-fold lower compared to the FH concentration in the same serum samples and FHR-3 did not behave as a major acute phase response protein.

The production and secretion of complement component C1q by human mast cells.

C1q is the initiation molecule of the classical pathway of the complement system and is produced by macrophages and immature dendritic cells. As mast cells share the same myeloid progenitor cells, we have studied whether also mast cells can produce and secrete C1q. Mast cells were generated in vitro from CD34+ progenitor cells from buffy coats or cord blood. Fully differentiated mast cells were shown by both RNA sequencing and qPCR to express C1QA, C1QB and C1QC. C1q produced by mast cells has a similar molecular make-up as serum C1q. Reconstituting C1q depleted serum with mast cell supernatant in haemolytic assays, indicated that C1q secreted by mast cells is functionally active. The level of C1q in supernatants produced under basal conditions was considerably enhanced upon stimulation with LPS, dexamethasone in combination with IFN- γ or via FcεRI triggering. Mast cells in human tissues stained positive for C1q in both healthy and in inflamed tissue. Moreover, mast cells in healthy and diseased skin appear to be the predominant C1q positive cells. Together, our data reveal that mast cells are able to produce and secrete functional active C1q and indicate mast cells as a local source of C1q in human tissue.