Complement Receptor Type 1 (CR1, CD35), the Inhibitor of BCR-Mediated Human B Cell Activation, Differentially Regulates TLR7, and TLR9 Induced Responses.

The complement system and Toll-like receptors (TLRs) are essential contributors of innate immunity. Separate activation of these systems has been shown to play a role in initiating and shaping the adaptive immune response, however the modulation of various B cell functions by the simultaneous involvement of these two systems has not yet been uncovered. We demonstrate here that occupancy of complement receptor type 1 (CR1, CD35) by its natural, complement component C3-derived ligand significantly and dose dependently reduces the TLR9-induced expression of activation markers, cytokine production, proliferation, and antibody production by human B cells, but has no effect on the TLR7-induced functions. The synergistic response to the simultaneous engagement of either TLR9 or TLR7 along with the BCR however, is significantly inhibited by CR1 occupancy. Our findings imply that both under physiological and pathological conditions, when complement- and TLR-activating microbial and damage products are present in the B cell environment, the cooperation between CR1 and TLR7 or TLR9 provides additional levels of the regulation of human B cell functions.

Non-specific adsorption of complement proteins affects complement activation pathways of gold nanomaterials.

The complement system is a key humoral component of innate immunity, serving as the first line of defense against intruders, including foreign synthetic nanomaterials. Although gold nanomaterials (AuNMs) are widely used in nanomedicine, their immunological response is not well understood. Using AuNMs of three shapes commonly used in biomedical applications: spherical gold nanoparticles, gold nanostars and gold nanorods, we demonstrated that AuNMs activated whole complement system, leading to the formation of SC5b-9 complex. All three complement pathways were simultaneously activated by all the AuNMs. Recognition molecules of the complement system interacted with all AuNMs in vitro, except for l-ficolin, but the correlation between these interactions and corresponding complement pathway activation was only observed in the classical and alternative pathways. We also observed the mediating role of complement activation in cellular uptake of all AuNMs by human U937 promonocytic cells, which expresses complement receptors. Taken together, our results highlighted the potential immunological challenges for clinical applications of AuNMs that were often overlooked.

Quantification of complement system activation by measuring C5b-9 cell surface deposition using a cell-ELISA technique.

The complement system is an important aspect of immune defense against microbial invasion. Eukaryotic cells express various complement regulatory proteins to protect them from uncontrolled complement activation. However, some eukaryotic cells possess constitutive complement system activation that does not require specific triggering factors, which is known to have unexpected effects on cell proliferation and survival. This area of research is still preliminary and a standard method to measure complement system activation in eukaryotic cells has yet to be identified. Here, we present a quantitative in vitro method to measure complement system activation in eukaryotic cells by detecting C5b-9, the membrane attack complex, on cell surfaces. The results obtained using this assay correlated with C3b deposition measured using flow cytometry and C5b-9 deposition detected using an immunofluorescence assay. Furthermore, we showed that various cancer cell lines displayed different levels of complement system activation by using this assay.

Ras-related GTPases Rap1 and RhoA collectively induce the phagocytosis of serum-opsonized zymosan particles in macrophages.

Phagocytosis occurs primarily through two main processes in macrophages: the Fcγ receptor- and the integrin αMß2-mediated processes. Complement C3bi-opsonized particles are known to be engulfed through integrin αMß2-mediated process, which is regulated by RhoA GTPase. C3 toxin fused with Tat-peptide (Tat-C3 toxin), an inhibitor of the Rho GTPases, was shown to markedly inhibit the phagocytosis of serum (C3bi)-opsonized zymosans (SOZs). However, 8CPT-2Me-cAMP, an activator of exchange protein directly activated by cAMP (Epac, Rap1 guanine nucleotide exchange factor), restored the phagocytosis of the SOZs that was previously inhibited by the Tat-C3 toxin. In addition, a constitutively active form of Rap1 GTPase (CA-Rap1) also restored the phagocytosis that was previously reduced by a dominant negative form of RhoA GTPase (DN-RhoA). This suggests that Rap1 can replace the function of RhoA in the phagocytosis. Inversely, CA-RhoA rescued the phagocytosis that was suppressed by DN-Rap1. These findings suggest that both RhoA and Rap1 GTPases collectively regulate the phagocytosis of SOZs. In addition, filamentous actin was reduced by the Tat-C3 toxin, which was again restored by 8CPT-2Me-cAMP. Small interfering profilin suppressed the phagocytosis, suggesting that profilin is essential for the phagocytosis of SOZs. Furthermore, 8CPT-2Me-cAMP increased the co-immunoprecipitation of profilin with Rap1, whereas Tat-C3 toxin decreased that of profilin with RhoA. Co-immunoprecipitations of profilin with actin, Rap1, and RhoA GTPases were augmented in the presence of GTPγS rather than GDP. Therefore, we propose that both Rap1 and RhoA GTPases regulate the formation of filamentous actin through the interaction between actin and profilin, thereby collectively inducing the phagocytosis of SOZs in macrophages.

Localised PtdIns(3,4,5)P3 or PtdIns(3,4)P2 at the phagocytic cup is required for both phagosome closure and Ca2+ signalling in HL60 neutrophils.

Several events accompany integrin-mediated phagocytosis by myeloid cells. These include local pseudopod and phagocytic cup formation followed by Ca(2+) signalling. However, there is also a role for localised phosphatidylinositol (3,4,5) trisphosphate [PtdIns(3,4,5)P(3)] production. Here we report that in neutrophilic HL-60 cells expressing PH-Akt-GFP, binding of iC3b-coated zymosan particles (2 microm in diameter) via beta2 integrin induces an incomplete phagocytic cup to form before either PtdIns(3,4,5)P(3) or phosphatidylinositol (3,4) bisphosphate [PtdIns(3,4)P(2)] production or Ca(2+) signalling. These phosphoinositides then accumulated locally at the site of the phagocytic cup and Ca(2+) signalling and phagosome closure follows immediately. Although photobleaching showed that PH-Akt-GFP was freely diffusible in the cytosol and able to dissociate from the phagocytic cup, it was restricted to the plasma membrane of the formed but open phagosome and failed to diffuse into the surrounding plasma membrane or neighbouring phagocytic cups even if connected. Inhibition of phosphoinositide (PI) 3-kinase or depletion of membrane cholesterol inhibited both Ca(2+) signalling and phagosome closure, but had no effect on particle binding or phagocytic cup formation. We therefore conclude that PtdIns(3,4,5)P(3) or PtdIns(3,4)P(2) generation was not required for the events that initiate the formation of the phagocytic cup, but that anchoring of PtdIns(3,4,5)P(3) at the phagocytic cup is an essential step for phagosome closure and Ca(2+) signalling.

A new apoptotic pathway for the complement factor B-derived fragment Bb.

Apoptosis is involved in both the cellular and humoral immune system destroying tumors. An apoptosis-inducing factor from HL-60 myeloid leukemia cells was obtained, purified, and sequenced. The protein found has been identified as a human complement factor B-derived fragment Bb, although it is known that factor B is able to induce apoptosis in several leukemia cell lines. Monoclonal antibodies against fragment Ba and Bb inhibited the apoptotic activity of factor B. When the purified fragment Bb was used for apoptosis induction, only the anti-Bb antibody inhibited Bb-induced apoptosis, and not the anti-Ba antibody. The apoptosis-inducing activity was found to be enhanced under conditions facilitating the formation of Bb. Blocking TNF/TNFR or FasL/Fas interactions did not interfere with the factor B-induced apoptosis. CD11c (iC3bR) acts as the main subunit of a heterodimer binding to fragment Bb in the apoptosis pathway, and the factor B-derived fragment Bb was found to possess the previously unknown function of inducing apoptosis in leukemic cells through a suicide mechanism of myeloid lineage cells during the differentiation stage.

The influence of IL-3, IL-5, and GM-CSF on normal human eosinophil and neutrophil C3b-induced degranulation.

The priming effect of interleukin-3 (IL-3), interleukin-5 (IL-5), and granulocyte/macrophage-colony stimulating factor (GM-CSF) on eosinophil and neutrophil degranulation was studied. Granulocytes were obtained from normal donors, and degranulation was induced by incubation with serum-opsonized Sephadex particles. The released amounts of eosinophil cationic protein (ECP), eosinophil protein X (EPX), myeloperoxidase (MPO), and lactoferrin (LF) were measured by radioimmunoassay (RIA). The effect of IL-5 was dose- and time-dependent, with a maximal enhancement of ECP and EPX release of 71% (P < 0.03) and 66% (P < 0.03), respectively. Neutrophil degranulation, however, was unaffected. IL-3 was marginally effective, whereas GM-CSF seemed to act as a secretagogue for both eosinophil and neutrophil degranulation. We conclude that IL-5 selectively primes eosinophil degranulation, whereas IL-3 and GM-CSF seem to act as secretagogues for eosinophils and neutrophils. The results indicate that IL-5 may be involved in the priming of eosinophils as observed in patients with asthma and hypereosinophilic syndrome (HES).

Kinetics of the biosynthesis of complement subcomponent C1q by murine macrophages: LPS, immune complexes, and zymosan alone and in combination with interferon-gamma.

We investigated the effects of bacterial lipopolysaccharide (LPS), immune complexes (IC), and C3b opsonized zymosan (AZ) alone and in combination with interferon-gamma (IFN-gamma) priming on macrophage synthesis and secretion of C1q. Our results indicated that LPS, IC, and AZ alone stimulated C1q mRNA and secretion in the absence of IFN-gamma. The increase in mRNA accumulation was detectable after 3 h, peaked at 6 h and was maintained at constitutive levels for 24 h. There was a corresponding early burst of increased secretion of functional C1q after 3 to 6 h which declined rapidly after 9 to 24 h culture of LPS-stimulated macrophages. Priming of macrophages with IFN-gamma and simultaneous triggering with LPS, IC, or AZ produced additive rather than synergistic increases in C1q mRNA accumulation. These same agents inhibited constitutive secretion of C1q in the absence of IFN-gamma priming as determined by autoradiographic analysis of metabolically radiolabeled secretory C1q. Triggering of IFN-gamma primed macrophages with LPS, IC, or AZ also markedly suppressed the increased rate of C1q secretion induced by IFN-gamma in a dose-related fashion. A corresponding dose-dependent increased accumulation of endogenous C1q in cell lysates was detected by Western blot analysis of macrophages which had been stimulated by LPS, IC, or AZ alone or in combination with IFN-gamma. Our findings indicate that LPS as well as FcR and C3bR triggering agents stimulate early and sustained C1q synthesis accompanied by an early and short-lived burst of C1q secretion which rapidly diminished and results in an increased intracellular accumulation of C1q due to ongoing synthesis. IFN-gamma appeared to further amplify the same kinetics of increased C1q mRNA accumulation and decreased extracellular accumulation mediated by LPS, IC, and ZM. Our results suggest that LPS, IC, and AZ alone or in combination with IFN-gamma stimulate early C1q production to modulate macrophage effector functions followed by an inhibition of C1q secretion when the activation process has been culminated.

Inhibition of lymphocyte proliferation by fluid-phase C3b as influenced by macrophages.

The effect of fluid-phase C3b on mitogen-induced lymphocyte proliferation in the presence and absence of macrophages was studied. In general, C3b inhibited the proliferation of lymphocytes when monocytes (macrophages) were present. The degree of inhibition by C3b was different for B and T lymphocytes and varied for different subpopulations of lymphocyte classes. In the absence of monocytes (macrophages), there was insignificant inhibition by C3b of lymphocyte proliferation, and thus the observed inhibition appeared to be due to the effect of C3b on the monocytes/macrophages present in the mixed lymphocyte preparations.

Substances that can trigger activation of the alternative pathway of complement have anti-melanoma activity in mice.

Intraperitoneal (i.p.) injection of substances that can ignite the alternative pathway of complement, namely isolated human C3b or C3(H2O), guinea-pig C3(H2O) or cobra venom factor, or conventionally prepared zymosan, will reproducibly and very significantly increase the mean survival time of C57BL mice previously inoculated i.p. with melanoma cells. The effect is greater at higher doses and earlier post-inoculation (p.i.) administration, but the substances are active at low doses (30-100 micrograms/mouse) if given early enough. It is likely that C3b or C3(H2O) was the previously unidentified anti-tumour factor activated in serum by S. aureus treatment or serum fractionation and described elsewhere. Activation of the alternative pathway of complement appears to have potential interest for cancer therapy.