Ngaisah Klar-Mohamad

Functional characterization of the lectin pathway of complement in human serum

Mannan-binding lectin (MBL) is a major initiator of the lectin pathway (LP) of complement. Polymorphisms in exon 1 of the MBL gene are associated with impaired MBL function and infections. Functional assays to assess the activity of the classical pathway (CP) and the alternative pathway (AP) of complement in serum are broadly used in patient diagnostics. We have now developed a functional LP assay that enables the specific quantification of autologous MBL-dependent complement activation in human serum. Complement activation was assessed by ELISA using coated mannan to assess the LP and coated IgM to assess the CP. Normal human serum (NHS) contains IgG, IgA and IgM antibodies against mannan, as shown by ELISA. These antibodies are likely to induce CP activation. Using C1q-blocking and MBL-blocking mAb, it was confirmed that both the LP and the CP contribute to complement activation by mannan. In order to quantify LP activity without interference of the CP, LP activity was measured in serum in the presence of C1q-blocking Ab. Activation of serum on coated IgM via the CP resulted in a dose-dependent deposition of C1q, C4, C3, and C5b-9. This activation and subsequent complement deposition was completely inhibited by the C1q-blocking mAb 2204 and by polyclonal Fab anti-C1q Ab. Evaluation of the LP in the presence of mAb 2204 showed a strong dose-dependent deposition of C4, C3, and C5b-9 using serum from MBL-wildtype (AA) but not MBL-mutant donors (AB or BB genotype), indicating that complement activation under these conditions is MBL-dependent and C1q-independent. Donors with different MBL genotypes were identified using a newly developed oligonucleotide ligation assay (OLA) for detection of MBL exon 1 polymorphisms. We describe a novel functional assay that enables quantification of autologous complement activation via the LP in full human serum up to the formation of the membrane attack complex. This assay offers novel possibilities for patient diagnostics as well as for the study of disease association with the LP.

FcαRI/CD89 Circulates in Human Serum Covalently Linked to IgA in a Polymeric State

The FcR for IgA CD89/FcαRI, is a type I receptor glycoprotein, expressed on myeloid cells, with important immune effector functions. In vitro CD89 can be released from CD89-expressing cells upon activation. Little information is available on the existence of this soluble molecule in vivo. Using specific and sensitive ELISA techniques (detection limit 50 pg/ml), we were not able to detect circulating CD89 in human sera. However, using Western blotting, a 30-kDa soluble CD89 molecule was demonstrated in both serum and plasma. Moreover, using a specific semiquantitative dot-blot system, we found CD89 in all human sera tested (mean concentration 1900 ng/ml). Size fractionation of human serum using gel filtration chromatography showed that the CD89 molecule was predominantly present in larger molecular mass fractions. Direct complexes between IgA and CD89 were demonstrated by anti-IgA affinity purification, and when analyzed under nonreducing conditions appeared to be covalently linked. Size fractionation of affinity-purified IgA showed the presence of soluble CD89 only in the high molecular mass fractions of IgA, but not in monomeric IgA. High molecular mass complexes of CD89-IgA could be distinguished from J chain containing dimeric IgA. These data show that CD89 circulates in complex with IgA, and suggest that CD89 might contribute to the formation of polymeric serum IgA.

Functional assessment of mouse complement pathway activities and quantification of C3b/C3c/iC3b in an experimental model of mouse renal ischaemia/reperfusion injury.

The complement system is an essential component of our innate immunity, both for the protection against infections and for proper handling of dying cells. However, the complement system can also contribute to tissue injury and inflammatory responses. In view of novel therapeutic possibilities, there is an increasing interest in measurement of the complement system activation in the systemic compartment, both in the clinical setting as well as in experimental models. Here we describe in parallel a sensitive and specific sandwich ELISA detecting mouse C3 activation fragments C3b/C3c/iC3b, as well as functional complement ELISAs detecting specific activities of the three complement pathways at the level of C3 and at the level of C9 activation. In a murine model of renal ischaemia/reperfusion injury (IRI) we found transient complement activation as shown by generation of C3b/C3c/iC3b fragments at 24 h following reperfusion, which returned to base-line at 3 and 7 days post reperfusion. When the pathway specific complement activities were measured at the level of C3 activation, we found no significant reduction in any of the pathways. However, the functional complement activity of all three pathways was significantly reduced when measured at the level of C9, with the strongest reduction being observed in the alternative pathway. For all three pathways there was a strong correlation between the amount of C3 fragments and the reduction in functional complement activity. Moreover, at 24 h both C3 fragments and the functional complement activities showed a correlation with the rise in serum creatinine. Together our results show that determination of the systemic pathway specific complement activity is feasible in experimental mouse models and that they are useful in understanding complement activation and inhibition in vivo.

Properdin and factor H production by human dendritic cells modulates their T‐cell stimulatory capacity and is regulated by IFN‐γ

Dendritic cells (DCs) and complement are both key members of the innate and adaptive immune response. Recent experimental mouse models have shown that production of alternative pathway (AP) components by DCs strongly affects their ability to activate and regulate T‐cell responses. In this study we investigated the production and regulation of properdin (fP) and factor H (fH) both integral regulators of the AP, by DCs and tolerogenic DCs (tolDCs). Both fP and fH were produced by DCs, with significantly higher levels of both AP components produced by tolDCs. Upon activation with IFN‐γ both cells increased fH production, while simultaneously decreasing production of fP. IL‐27, a member of the IL‐12 family, increased fH, but production of fP remained unaffected. The functional capacity of fP and fH produced by DCs and tolDCs was confirmed by their ability to bind C3b. Inhibition of fH production by DCs resulted in a greater ability to induce allogenic CD4+ T‐cell proliferation. In contrast, inhibition of fP production led to a significantly reduced allostimulatory capacity. In summary, this study shows that production of fP and fH by DCs, differentially regulates their immunogenicity, and that the local cytokine environment can profoundly affect the production of fP and fH.

Binding and degradation of soluble immunoglobulin aggregates by mouse mononuclear phagocytes--stimulation by colony-stimulating factor.

nduced peritoneal macrophages bound and degraded the aggregates to the same degree as the cultured bone marrow mononuclear phagocytes did. However, the thioglycollate-i nduced macrophages lost most of these capacities when cultured in vitro without CM. When CM was added to these cultures, the capacity to bind and degrade was restored in a dose-dependent fashion. To obtain the maximum effect, exposure to CM must be maintained for more than 2 days. The effect of CM could be reproduced with purified CSF-1. Taken together the results of this study indicate that Fc receptor expression is modulated by CSF- 1 .

Systemic complement activation in central serous chorioretinopathy

Purpose A clear link between several variants in genes involved in the complement system and chronic central serous chorioretinopathy (CSC) has been described. In age-related macular degeneration, a disease that shows clinical features that overlap with CSC, both genetic risk factors and systemic activation of the complement system have previously been found. In this case-control study, we assessed whether there is evidence of either systemic activation or inhibition of the complement system in patients with chronic CSC. Methods A prospective case-control study of 76 typical chronic CSC patients and 29 controls without ophthalmological history was conducted. Complement activity assays (classical, alternative, and mannose-binding lectin pathway), complement factors 3, 4, 4A, 4B, B, D, H, I, and P, activation products C3d, C5a, and sC5b-C9, and the C3d/C3 ratio were analysed in either serum or plasma. A correction for possible effects of gender, age, body mass index, and smoking status was performed. Results In this study, none of the tested variables, including regulation and activation products, proved to be significantly different between the groups. Moreover, no associations with either CSC disease activity or possible CSC related steroid use were observed. Conclusion Despite the available literature regarding a possible relationship between chronic CSC and variants in genes involved in the complement system, we did not find evidence of an association of chronic CSC with either systemic complement activation or inhibition.