Yersinia pestis Δail Mutants Are Not Susceptible to Human Complement Bactericidal Activity in the Flea.

Ail confers serum resistance in humans and is a critical virulence factor of Y. pestis, the causative agent of plague. Here, the contribution of Ail for Y. pestis survival in the flea vector was examined. Rat or human but not mouse sera were bactericidal against a Y. pestis Δail mutant at 28°C in vitro. Complement components deposited rapidly on the Y. pestis surface as measured by immunofluorescent microscopy. Ail reduced the amount of active C3b on the Y. pestis surface. Human sera retained bactericidal activity against a Y. pestis Δail mutant in the presence of mouse sera. However, in the flea vector, the serum protective properties of Ail were not required. Flea colonization studies using murine sera and Y. pestis KIM6+ wild type, a Δail mutant, and the Δail/ail+ control showed no differences in bacterial prevalence or numbers during the early stage of flea colonization. Similarly, flea studies with human blood showed Ail was not required for serum resistance. Finally, a variant of Ail (AilF100V E108_S109insS) from a human serum-sensitive Y. pestis subsp. microtus bv. Caucasica 1146 conferred resistance to human complement when expressed in the Y. pestis KIM6+ Δail mutant. This indicated that Ail activity was somehow blocked, most likely by lipooligosaccharide, in this serum sensitive strain. IMPORTANCE This work contributes to our understanding of how highly virulent Y. pestis evolved from its innocuous enteric predecessor. Among identified virulence factors is the attachment invasion locus protein, Ail, that is required to protect Y. pestis from serum complement in all mammals tested except mice. Murine sera is not bactericidal. In this study, we asked, is bactericidal sera from humans active in Y. pestis colonized fleas? We found it was not. The importance of this observation is that it identifies a protective niche for the growth of serum sensitive and nonsensitive Y. pestis strains.

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.

The secreted Candida albicans protein Pra1 disrupts host defense by broadly targeting and blocking complement C3 and C3 activation fragments.

Candida albicans the most frequently isolated clinical fungal pathogen can cause local as well as systemic and life-threatening infections particularly in immune-compromised individuals. A better and more detailed understanding how C. albicans evades human immune attack is therefore needed for identifying fungal immune-evasive proteins and develop new therapies. Here, we identified Pra1, the pH-regulated C. albicans antigen as a hierarchical complement inhibitor that targets C3, the central human complement component. Pra1 cleaved C3 at a unique site and further inhibited effector function of the activation fragments. The newly formed C3a-like peptide lacked the C-terminal arginine residue needed for C3a-receptor binding and activation. Moreover, Pra1 also blocked C3a-like antifungal activity as shown in survival assays, and the C3b-like molecule formed by Pra1 was degraded by the host protease Factor I. Pra1 also bound to C3a and C3b generated by human convertases and blocked their effector functions, like C3a antifungal activity shown by fungal survival, blocked C3a binding to human C3a receptor-expressing HEK cells, activation of Fura2-AM loaded cells, intracellular Ca2+ signaling, IL-8 release, C3b deposition, as well as opsonophagocytosis and killing by human neutrophils. Thus, upon infection C. albicans uses Pra1 to destroy C3 and to disrupt host complement attack. In conclusion, candida Pra1 represents the first fungal C3-cleaving protease identified and functions as a fungal master regulator of innate immunity and as a central fungal immune-escape protein.

FHR-1 Binds to C-Reactive Protein and Enhances Rather than Inhibits Complement Activation.

Factor H-related protein (FHR) 1 is one of the five human FHRs that share sequence and structural homology with the alternative pathway complement inhibitor FH. Genetic studies on disease associations and functional analyses indicate that FHR-1 enhances complement activation by competitive inhibition of FH binding to some surfaces and immune proteins. We have recently shown that FHR-1 binds to pentraxin 3. In this study, our aim was to investigate whether FHR-1 binds to another pentraxin, C-reactive protein (CRP), analyze the functional relevance of this interaction, and study the role of FHR-1 in complement activation and regulation. FHR-1 did not bind to native, pentameric CRP, but it bound strongly to monomeric CRP via its C-terminal domains. FHR-1 at high concentration competed with FH for CRP binding, indicating possible complement deregulation also on this ligand. FHR-1 did not inhibit regulation of solid-phase C3 convertase by FH and did not inhibit terminal complement complex formation induced by zymosan. On the contrary, by binding C3b, FHR-1 allowed C3 convertase formation and thereby enhanced complement activation. FHR-1/CRP interactions increased complement activation via the classical and alternative pathways on surfaces such as the extracellular matrix and necrotic cells. Altogether, these results identify CRP as a ligand for FHR-1 and suggest that FHR-1 enhances, rather than inhibits, complement activation, which may explain the protective effect of FHR-1 deficiency in age-related macular degeneration.

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.

Macrolides and ß-lactam antibiotics enhance C3b deposition on the surface of multidrug-resistant Streptococcus pneumoniae strains by a LytA autolysin-dependent mechanism.

The emergence of Streptococcus pneumoniae strains displaying high levels of multidrug resistance is of great concern worldwide and a serious threat for the outcome of the infection. Modifications of the bacterial envelope by antibiotics may assist the recognition and clearance of the pathogen by the host immune system. Recognition of S. pneumoniae resistant strains by the complement component C3b was increased in the presence of specific anti-pneumococcal antibodies and subinhibitory concentrations of different macrolides and ß-lactam antibiotics for all the strains investigated. However, C3b levels were unchanged in the presence of serum containing specific antibodies and sub-MICs of levofloxacin. To investigate whether LytA, the main cell wall hydrolase of S. pneumoniae, might be involved in this process, lytA-deficient mutants were constructed. In the presence of antibiotics, loss of LytA was not associated with enhanced C3b deposition on the pneumococcal surface, which confirms the importance of LytA in this interaction. The results of this study offer new insights into the development of novel therapeutic strategies using certain antibiotics by increasing the efficacy of the host immune response to efficiently recognize pneumococcal resistant strains.

[The effects of complement C3f segment on expression and secretion of collagen I, III and transforming growth factor-beta1 in human embryonic lung fibroblast].

OBJECTIVE: To observe the effects of complement fragment C3f on expression and secretion of collagen I, III and transforming growth factor( TGF)-beta1 in human embryonic lung fibroblast (MRC-5) cells. METHODS: MRC-5 cells were cultured with C3f (the synthetic 17 peptides fragments of complement C3). The extracellular and intracellular expression levels of type I, III collagens and TGF-beta1 in MRC-5 cultures were detected by ELISA and immunohistochemistry, respectively. RESULTS: The expression levels of type I, III collagen and TGF-beta1 in the supernatant of MRC-5 cultures decreased significantly with the concentrations of C3f as compared with controls (P < 0.05). Also the expression level of TGF-beta1 in MRC-5 cytoplasm reduced significantly as compared with controls (P < 0.05). CONCLUSION: The results of present in vitro study showed that the complement fragment C3f could reduce the formation of TGF-beta1 and type I, III collagens in MRC-5 cells, and inhibit the lung tissue fibrosis.

Reversal of iC3b-inhibited dendritic cell differentiation via inhibition of the extracellular signal-regulated mitogen-activated protein kinase promotes CD4(+) T cell proliferation.

OBJECTIVES: To investigate the roles of ERK1/2 and p38 MAPK cascades in the differentiation of iC3b-combined CD14(+) monocyte into CD1a(+) MDDC, and to study how these cells influence CD4(+) T cell proliferation. METHODS: CD14(+) monocyte was co-cultured with iC3b with or without inhibitors specific for ERK1/2 or p38 MAPK pathways for 2days, then the expressions of CD14, CD1a, phophso-ERK1/2, phophso-p38, IL-10 and IL-12 p70 were detected, and CD4(+) T cell proliferation was measured via (3)H-TdR as well. RESULTS: Maturation of CD1a(+) DC was inhibited by iC3b along with downregulated expressions of CD1a, phophso-p38 and IL-12p70 and upregulated expressions of phophso-ERK1/2 and IL-10, and the CD4(+) T cell proliferation was restrained accordingly. When pretreated with inhibitor specific for ERK1/2 pathway, the inhibited maturation of imDC was reversed prominently with a higher level expression of CD1a and IL-12p70, whereas expressions of phophso-ERK1/2 and IL-10 were lowered, and accordingly the CD4(+) T cell proliferation restored significantly. CONCLUSIONS: iC3b inhibited the differentiation of CD14(+) monocytes into CD1a(+) MDDCs via ERK1/2 pathway, and restoration of CD1a(+) MDDCs maturation occurred with the treatment of inhibitors specific for ERK1/2 pathway. Meanwhile, treatment of the inhibitor for the ERK1/2 cascade reversed the inhibited CD4(+) T cell proliferation, implying a potential possibility for clinical intervention.

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.

Effects of complement component 3 derivatives on pig oocyte maturation, fertilization and early embryo development in vitro.

Complement component 3 (C3) has well-established roles within immune system, but its roles outside of immune system are less characterized. The extensive presence of C3 throughout the female reproductive tract, and its temporal, and gamete-specific regulation of expression suggest a potential role for C3 in reproduction. In the present investigation, the effects of C3, C3b and iC3b on porcine oocyte maturation, fertilization and embryonic development were examined. We identified the ability of iC3b to positively influence oocyte maturation. No effects on fertilization efficiency, penetration rates, polyspermy and blastocyst formation were observed. However, C3, C3b and iC3b presence in embryo culture medium resulted in fewer total cells in test blastocysts compared to control blastocysts. The results of this study indicate a potential function for iC3b in oocyte maturation. Furthermore, it was demonstrated that the presence of either C3, C3b or iC3b has a negative influence on early embryonic development in the porcine species.

Complement component C3 functions as an embryotrophic factor in early postimplantation rat embryos.

A presumed embryotrophic factor for early postimplantation rat embryos, partially purified from rat serum, was identified as complement component C3 (C3), the central component of the complement system, by sequence analysis of its N-terminal. Purified rat C3 showed embryotrophic activity for rat embryos cultured from day 9.5 of gestation for 48 h in the culture medium composed of rabbit serum. The maximum embryotrophic activity of C3 was observed around 0.5 mg/ml, a level which is lower than rat serum C3 levels. In the culture medium composed of rat serum, cultured rat embryos selectively consumed C3, and C3-depletion by cobra venom factor affected embryonic growth. Inactivation of the internal thiolester bond of C3, the critical functional site for its activity in the complement system, by methylamine had no effects on its embryotrophic activity. Purified rabbit C3 had only weak embryotrophic activity for cultured rat embryos, suggesting species specificity of the embryotrophic activity of C3. Immunochemical analyses showed the specific presence of C3 on the visceral yolk sac, but not on the embryo proper of day 9.5 or 10.5 rat embryos both in utero and in vitro. In analysis using fluorescein-labeled rat C3, unfragmented C3s bound to the visceral yolk sac stronger than C3b, the primary active fragment of C3 in the complement system. These results indicate that C3, which has always been considered to be detrimental to embryos, functions as an embryotrophic factor by novel mechanisms probably through the visceral yolk sac. The present study thus provides new insights into functions of C3 and postimplantation embryonic growth.

Cutting edge: Complement (C3d)-linked antigens break B cell anergy.

C3dg adducts of Ag can coligate complement receptor type 2 (CR2; CD21) and the B cell Ag receptor. This interaction significantly amplifies BCR-mediated signals in Ag-naive wild-type mice, lowering the threshold for B cell activation and the generation of humoral immune responses as much as 1000-fold. In this study we demonstrate that CR2-mediated complementation of BCR signals can also overcome B cell anergy. Unlike Ag alone, BCR/CR2 costimulation (Ars-CCG/C3dg complexes) of anergic Ars/A1 B cells led to Ca(2+) mobilization in vitro and the production of autoantibodies in vivo. Interestingly, the in vivo immune response of anergic cells occurs without the formation of germinal centers. These results suggest that the Ag unresponsiveness of anergic B cells can be overcome by cross-reactive (self-mimicking) Ags that have been complement-opsonized. This mechanism may place individuals exposed to complement-fixing bacteria at risk for autoimmunity.

[The immune enhancement of the C3 to the Escherichia coli vaccine].

C3b was separated and purified from the SPF chicken serum. It was linked with E. coli antigen by the glutaral. 11 days aged SPF chicken were immunized by the complex antigen and the chickens of control group were immunised by the FCA- E. coli antigen . They were boosted at the age of 18 day. The immune response was monitored by an enzyme-linked immunosorbent assay(ELISA) for anti-E. coli anitibody. The ELISA results indicated that during the early several weeks, IgG titers elicited by FCA (FCA-E. coli) were higher than those elicited by C3 (C3b-E. coli), but decreased rapidly after a peak around the end of 4th week from being immunized. Chickens immunized with C3b always gave increased response, and the IgG titers were equal to that of FCA at the end of 5th week from being immunized and then higher and higher than that of FCA. Thus the adjuvant effect of C3b is different from that of FCA, it could induce production of memory cell and make the antigens stimulate immune cells consistently and stably.

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.

The human complement regulator factor H binds pneumococcal surface protein PspC via short consensus repeats 13 to 15.

The innate ability of Streptococcus pneumoniae to resist complement activation and complement-mediated phagocytosis may be a direct consequence of the ability of the bacteria to bind components of the complement regulatory system. One such component, factor H (fH), is a crucial fluid-phase negative regulator of the alternative pathway of complement and is utilized by a number of pathogenic organisms to resist complement attack. The pneumococcal surface protein C (PspC [also known as CbpA] and SpsA) has been shown to bind fH, although the exact binding site within one or more of the 20 short consensus repeats (SCRs) of the molecule is not known. The purpose of the current study was to map specific SCRs on fH responsible for this binding. Initial experiments utilizing type 2 pneumococcal strain D39 and its isogenic PspC-negative derivative (D39/pspC mutant) showed that fH binding was PspC dependent. A purified recombinant protein derivative of PspC that lacked the proline-rich region (PspCDeltaPro) had a reduced binding efficiency for fH, thereby directly showing the importance of this region for the fH interaction. We have specifically shown by inhibition experiments that SCRs responsible for heparin and C3b binding of fH are not involved in binding PspC and the interaction between fH and PspC is largely hydrophobic, since no inhibition was observed in the presence of high concentrations of NaCl. Construction of SCR proteins encompassing the whole fH molecule showed that SCRs 8 to 15 (SCR 8-15) mediated binding to PspC. Further localization experiments revealed that SCR 13 and SCR 15 were required for full binding, although partial binding was retained when either SCR was removed.

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.

Replenishment of RANTES mRNA expression in activated eosinophils from atopic asthmatics.

Eosinophils have been shown to express the gene encoding regulated upon activation, normal T-cell expressed and secreted (RANTES), a potent eosinophilotactic chemokine. RANTES protein expression in eosinophils has previously been shown to be up-regulated by a number of agonists, including complement-dependent factors (C3b/iC3b) and interferon-gamma (IFN-gamma). We hypothesized that gene expression of RANTES is regulated in these cells by eosinophil-specific agonists. We analysed RANTES mRNA expression by reverse-transcription polymerase chain reaction (RT-PCR) in human peripheral blood eosinophils obtained from mild atopic asthmatics following stimulation over time. In resting eosinophils, a low level of RANTES mRNA was found to be constitutively expressed in all the atopic donors tested in this study (n = 6). Following stimulation with C3b/iC3b (serum-coated surfaces), eosinophils released measurable levels of RANTES, while sustained transcript expression was detected for up to 24 hr of stimulation. In contrast, IFN-gamma (5 ng/ml) transiently and significantly (P<0.05, n = 3) depleted relative amounts of RANTES PCR product (compared with beta2-microglobulin) after 1-4 hr of stimulation. RANTES transcript was again detectable after 24 hr of IFN-gamma incubation, suggesting that the pool of RANTES mRNA had been replenished. Other eosinophil-active cytokines, interleukin-3 (IL-3), IL-4, IL-5 and granulocyte-macrophage colony-stimulating factor, did not appear to modulate RANTES mRNA expression after 1 hr of incubation. The effect of IFN-gamma on RANTES mRNA was reversed by cycloheximide, suggesting that IFN-gamma may act by increasing the rate of translation of RANTES mRNA. These findings indicate that IFN-gamma may induce a rapid and transient effect on the translation and replenishment of RANTES mRNA in eosinophils. This novel observation supports the notion that eosinophils have the potential to replenish their stored and released bioactive proteins.

Different stimulating effects of complement C3b and complete Freund's adjuvant on antibody response.

Upon activation, complement C3 undergoes a conformational change and acquires the capacity to covalently bind to other proteins such as antigen and to interact with specific receptors; therefore, C3 is involved in cell mediated immune response. The adjuvant effect produced by linking C3-fragments to antigen has recently been described. We injected C3b-Ag complexes consisting of one molecule of C3b ester linked to one molecule of HEL to immunised mice, and we compared the C3b adjuvant activity with that of complete Freund's adjuvant. IgG titers elicited by HEL emulsified in CFA (HEL + CFA) were higher than those elicited by HEL-C3b, but decreased rapidly after a peak response around day 45 whereas HEL-C3b resulted in a continuous increase of anti-HEL response. Mice immunised with HEL + CFA then boosted with HEL-C3b gave significantly higher response than those boosted with HEL + CFA, indicating more efficient memory cell restimulation by C3b. HEL + CFA leads to better priming than HEL-C3b when mice are boosted with HEL-C3b. Thus, adjuvant effect of C3b is different from that of CFA, leading to more stable IgG production and better memory stimulation.

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).