C3 Secretion Research Articles

Evaluation of chylomicron effect on ASP production in 3T3-L1 adipocytes

In the past few years, there has been increasing interest in the production and physiological role of acylation-stimulating protein (ASP), identical to C3adesArg, a product of the alternative complement pathway generated through C3 cleavage. Recent studies in C3 (-/-) mice that are ASP deficient have demonstrated a role for ASP in postprandial triglyceride clearance and fat storage. The aim of the present study was to establish a cell model and sensitive ELISA assay for the evaluation of ASP production using 3T3-L1 adipocytes. 3T3-L1 preadipocytes were differentiated into adipocytes, then cultured in different media such as serum-free (SF), Dulbecco's modified Eagle's medium (DMEM)/F12 + 10% fetal calf serum (FBS), and at varying concentrations of chylomicrons and insulin + chylomicrons up to 48 h. ASP production in SF and DMEM/F12 + 10% FBS was compared. Chylomicrons stimulated ASP production in a concentration- and time-dependent manner. By contrast, chylomicron treatment had no effect on the production of C3, the precursor protein of ASP, which was constant over 48 h. Addition of insulin (100 nM) to a low-dose of chylomicrons (100 µg TG/ml) significantly increased ASP production compared with chylomicrons alone at 48 h (P < 0.001). Furthermore, addition of insulin significantly increased C3 secretion at both 18 and 48 h of incubation (P < 0.05, P < 0.001, respectively). Overall, the proportion of ASP to C3 remained constant, indicating no change in the ratio of C3 cleaved to generate ASP. This study demonstrated that 3T3-L1 adipocyte is a useful model for the evaluation of C3 secretion and ASP production by using a sensitive mouse-specific ELISA assay. The stimulation of ASP production with chylomicrons demonstrates a physiologically relevant response, and provides a strategy for further studies on ASP production and function.

Biphasic expression and cytokine regulation of the complement C3 in heart allograft

Locally synthesized complement may play a more important role in regulating antigen-specific immune response than circulating complement; however, complement production in transplanted heart remains obscure. In the current study, we investigated local production of the complement C3 in mouse cardiac allografts and examined the relationship between C3 production and cytokine expression. The cardiac grafts of allogeneic (C57BL/6–BALB/c mice) and syngeneic mouse models were subjected to histopathological and immunohistochemical analyses for C3 expression on days 0–6 after operation. mRNA and protein expression of C3, IL-2, IFN-γ in transplanted heart and primary culture cardiomyocytes were analyzed. Our results demonstrated that C3 mRNA exhibited biphasic patterns in transplanted heart. The first expression phase correlated with ischemical reperfusion injury over 2days post-transplant. The second peak of C3 deposition was found only in allografts on day 5, concurrent with the secretion of IL-2 and IFN-γ accompanied by severe diffuse leukocyte infiltration. Furthermore, in vitro studies showed that IL-2 and IFN-γ enhanced C3 mRNA and protein production in cardiocytes. Together, these observations suggest that both ischemical reperfusion injury and the subsequent acute rejection result in elevated cardiocyte secretion of C3, and the second phase of expression appears to be regulated by cytokines secreted by the infiltrating cells.

Antagonistic Control of IgG-mediated Mast Cell Activation by Lyn and Fyn Kinases (86.8)

Abstract Mast cells are key players of the innate immune system. In addition to their role in atopic disease, mast cell involvement is strongly suspected in inflammatory diseases such as multiple sclerosis, rheumatoid arthritis, and atherosclerosis. Once activated, they degranulate and produce cytokines, resulting in the recruitment of other immune cells. It is well-appreciated that mast cells are activated via the high-affinity immunoglobulin E (IgE) receptor FcϵRI. In this study we focus on mast cell activation via the immunoglobulin G (IgG) receptor FcγRII/RIII and downstream signaling by Fyn and Lyn kinases. Lyn-deficient mast cells displayed increased FcγR expression, correlating with enhanced IgG-mediated histamine and leukotriene C4 secretion. The mast cells did not show a change in IgG-mediated cytokine production, suggesting a restricted and negative regulatory role for Lyn kinase in mast cell IgG signaling. In contrast, we show that Fyn kinase is rapidly activated by IgG crosslinkage, and is required for both IgG-mediated histamine release and cytokine production in mast cells. We obtained similar results when studying Lyn- or Fyn-deficient basophils and macrophages. Finally, we found that Lyn-deficient mice had exacerbated responses to passive systemic anaphylactic shock elicited by IgG challenge in vivo. These data illustrate the importance of Src family kinases in IgG signaling, a means by which mast cells may be activated in chronic inflammatory diseases.

The increased mucosal mRNA expressions of complement C3 and interleukin-17 in inflammatory bowel disease

Recent studies have demonstrated that the complement system participates in the regulation of T cell functions. To address the local biosynthesis of complement components in inflammatory bowel disease (IBD) mucosa, we investigated C3 and interleukin (IL)-17 mRNA expression in mucosal samples obtained from patients with IBD. The molecular mechanisms underlying C3 induction were investigated in human colonic subepithelial myofibroblasts (SEMFs). IL-17 and C3 mRNA expressions in the IBD mucosa were evaluated by real-time polymerase chain reaction. The C3 levels in the supernatant were determined by enzyme-linked immunosorbent assay. IL-17 and C3 mRNA expressions were elevated significantly in the active lesions from ulcerative colitis (UC) and Crohn's disease (CD) patients. There was a significant positive correlation between IL-17 and C3 mRNA expression in the IBD mucosa. IL-17 stimulated a dose- and time-dependent increase in C3 mRNA expression and C3 secretion in colonic SEMFs. The C3 molecules secreted by colonic SEMFs were a 115-kDa alpha-chain linked to a 70-kDa beta-chain by disulphide bonds, which was identical to serum C3. The IL-17-induced C3 mRNA expression was blocked by p42/44 mitogen-activated protein kinase (MAPK) inhibitors (PD98059 and U0216) and a p38 MAPK inhibitor (SB203580). Furthermore, IL-17-induced C3 mRNA expression was inhibited by an adenovirus containing a stable mutant form of I kappaB alpha. C3 and IL-17 mRNA expressions are enhanced, with a strong correlation, in the inflamed mucosa of IBD patients. Part of these clinical findings was considered to be mediated by the colonic SEMF response to IL-17.

Hormone and pharmaceutical regulation of ASP production in 3T3‐L1 adipocytes

Several studies have demonstrated increases in acylation stimulating protein (ASP), and precursor protein C3 in obesity, diabetes and dyslipidemia, however the nature of the regulation is unknown. To evaluate chronic hormonal and pharmaceutical mediated changes in ASP and potential mechanisms, 3T3-L1 adipocytes were treated with physiological concentrations of relevant hormones and drugs currently used in treatment of metabolic diseases for 48 h. Medium ASP production and C3 secretion were evaluated in relation to changes in adipocyte lipid metabolism (cellular triglyceride (TG) mass, non-esterified fatty acid (NEFA) release and real-time FA uptake). Chylomicrons increased ASP production (up to 411 +/- 133% P < 0.05), while leptin, triiodothyronine, and beta-blockers atenolol and propranolol had no effect. Dexamethasone, lovastatin, rosiglitazone and rimonabant decreased ASP production (-53 to -85%, P < 0.05), associated with a decrease in the precursor protein C3 (-37% to -65%, P < 0.01). By contrast, epinephrine, progesterone, testosterone, angiotensin II and metformin also decreased ASP (-54% to -100%, P < 0.05), but without change in precursor protein C3, suggesting a direct effect on convertase activity, possibly mediated by interference (except metformin) due to marked increases in NEFA (5.6-31-fold, increased P < 0.05). Both lovastatin and metformin induced decreases in ASP were also associated with decreased TG mass (maximal -60%, P < 0.05) and real-time FA uptake (maximum -75%, P < 0.05), suggesting a change in adipocyte differentiation status. These in vitro results are consistent with in vivo ASP profiles in subjects, and suggest that ASP may be regulated through precursor C3 availability, convertase activity and differentiation status.

STIM1 Is a Calcium Sensor Specialized for Digital Signaling

When cells are activated by calcium-mobilizing agonists at low, physiological concentrations, the resulting calcium signals generally take the form of repetitive regenerative discharges of stored calcium, termed calcium oscillations [1]. These intracellular calcium oscillations have long fascinated biologists as a mode of digitized intracellular signaling. Recent work has highlighted the role of calcium influx as an essential component of calcium oscillations [2]. This influx occurs through a process known as store-operated calcium entry, which is initiated by calcium sensor proteins, STIM1 and STIM2, in the endoplasmic reticulum [3]. STIM2 is activated by changes in endoplasmic reticulum calcium near the resting level, whereas a threshold of calcium depletion is required for STIM1 activation [4]. Here we show that, surprisingly, it is STIM1 and not STIM2 that is exclusively involved in calcium entry during calcium oscillations. The implication is that each oscillation produces a transient drop in endoplasmic reticulum calcium and that this drop is sufficient to transiently activate STIM1. This transient activation of STIM1 can be observed in some cells by total internal reflection fluorescence microscopy. This arrangement nicely provides a clearly defined and unambiguous signaling system, translating a digital calcium release signal into calcium influx that can signal to downstream effectors.

Opposing Roles for Lyn and Fyn Kinases in Mast Cell IgG Receptor Signaling (139.4)

Abstract Mast cells are key players of the innate immune system. In addition to their role in atopic disease, mast cell involvement is strongly suspected in inflammatory diseases such as multiple sclerosis, rheumatoid arthritis, and atherosclerosis. Once activated, they degranulate and produce cytokines, resulting in the recruitment of other immune cells. It is well-appreciated that mast cells are activated via the high-affinity immunoglobulin E (IgE) receptor FceRI. In this study we focus on mast cell activation via the immunoglobulin G (IgG) receptor FcγRII/RIII and downstream signaling by Fyn and Lyn kinases. Lyn-deficient mast cells displayed increased histamine and leukotriene C4 secretion, but no change in cytokine production, suggesting a restricted and negative regulatory role for Lyn kinase in mast cell IgG signaling. In contrast, we show that Fyn kinase is required for IgG-mediated histamine, LTC4, and cytokine production in mast cells, and has a similar role in basophils and macrophages. These data illustrate the importance of Src family kinases in IgG signaling, a means by which mast cells may be activated in chronic inflammatory diseases. Supported by NIH grants 1R01 AI59638 and U19A1077435.

Adiponectin is present in the urine in its native conformation, and specifically reduces the secretion of MCP‐1 by proximal tubular cells

To determine whether adiponectin detected in urine is present in its native form and if adiponectin receptors (AdipoR) present and functional in proximal tubular (HK-2) cells. Adiponectin is a protein with anti-inflammatory, anti-atherogenic and insulin-sensitizing properties. It has previously been detected antigenically in the urine in several forms of renal disease. We compared the isoform distribution of urinary adiponectin in patients with proteinuric and non-proteinuric renal disease with that of matched controls using chromatography and enzyme-linked immunosorbent assay. We examined whether AdipoR were present in HK-2 cells by real-time reverse transcription polymerase chain reaction. Their functionality was investigated by determining the effect of recombinant adiponectin on adenosine monophosphate-activated protein kinase phosphorylation using western blotting, and on the secretion of monocyte chemotactic protein-1 and C3 using enzyme-linked immunosorbent assays. Adiponectin in the urine is physiologically intact and largely present as the low molecular weight isoform. Subjects with urinary protein >150 mg/L excreted significantly more adiponectin and its high and low molecular weight isoforms than those with <150 mg/L. mRNA for AdipoR were present in HK-2 cells, with levels of mRNA for AdipoR1 being 20 times greater than those for AdipoR2. Ligation of AdipoR on proximal tubular cells increased phosphorylation of adenosine monophosphate-activated protein kinase, and downregulated the secretion of the inflammatory cytokine monocyte chemotactic protein-1, but not of C3. Physiologically relevant isoforms of adiponectin are present in the urine of normal subjects and those with proteinuria. In addition, functional receptors for adiponectin are present in HK-2 cells. Abnormal levels of adiponectin in the urine may therefore activate these receptors, potentially resulting in anti-inflammatory activity.

Regulation of complement‐3 protein expression in human and mouse oviducts

The human oviduct derived embryotrophic factor-3 (ETF-3) contains complement protein-3 (C3) and its derivates. Although C3 is not embryotrophic, it is converted into the embryotrophic derivative, iC3b in the presence of embryos and oviductal cells. The regulation of C3 production in the oviduct is not known. The objectives of this study were to investigate the effects of presence of preimplantation embryos and hormones on C3 expression in the oviducts in vitro and in vivo. The expression of C3 in the oviduct of pregnant mice was compared to that of pseudo-pregnant mice. The hormonal action on C3 expression was studied in the ovariectomized mouse oviducts and human oviductal epithelial (OE) cells. The results showed that the level of C3 mRNA in the mouse oviduct was high on Day 1 and Day 2, but decreased to a minimum on Day 4 of pregnancy, whereas that of pseudo-pregnancy remained relatively stable within the same period. The protein levels of C3 and iC3b specific fragments, alpha-115 and alpha-40, respectively in the mouse oviductal luminal fluid were highest on Day 3 of pregnancy, when the embryos were expected to be most sensitive to the embryotrophic activity of ETF-3. Estrogen elevated C3 expression in the ovariectomized mouse oviduct and the OE cells. Progesterone suppressed estrogen-induced C3 expression in the mouse oviduct, but had no effect on OE cells. In conclusion, the presence of embryo and steroid hormones regulate the synthesis and secretion of oviductal C3.

The Complement Inhibitor Low Molecular Weight Dextran Sulfate Prevents TLR4-Induced Phenotypic and Functional Maturation of Human Dendritic Cells

Low molecular weight dextran sulfate (DXS) has been reported to inhibit the classical, alternative pathway as well as the mannan-binding lectin pathway of the complement system. Furthermore, it acts as an endothelial cell protectant inhibiting complement-mediated endothelial cell damage. Endothelial cells are covered with a layer of heparan sulfate (HS), which is rapidly released under conditions of inflammation and tissue injury. Soluble HS induces maturation of dendritic cells (DC) via TLR4. In this study, we show the inhibitory effect of DXS on human DC maturation. DXS significantly prevents phenotypic maturation of monocyte-derived DC and peripheral myeloid DC by inhibiting the up-regulation of CD40, CD80, CD83, CD86, ICAM-1, and HLA-DR and down-regulates DC-SIGN in response to HS or exogenous TLR ligands. DXS also inhibits the functional maturation of DC as demonstrated by reduced T cell proliferation, and strongly impairs secretion of the proinflammatory mediators IL-1beta, IL-6, IL-12p70, and TNF-alpha. Exposure to DXS leads to a reduced production of the complement component C1q and a decreased phagocytic activity, whereas C3 secretion is increased. Moreover, DXS was found to inhibit phosphorylation of IkappaB-alpha and activation of NF-kappaB. These findings suggest that DXS prevents TLR-induced maturation of human DC and may therefore be a useful reagent to impede the link between innate and adaptive immunity.

Activation of the endothelium by IL-1 alpha and glucocorticoids results in major increase of complement C3 and factor B production and generation of C3a.

Constitutive secretion of complement C3 and factor B by the endothelial cell (EC) is lowered by therapeutic concentrations of glucocorticoids such as hydrocortisone or dexamethasone, whereas regulatory protein factor H production is increased by these hormones. In contrast, the proinflammatory cytokine IL-1 alpha has a stimulatory effect on C3 and factor B secretion by the endothelium and an inhibitory effect on factor H secretion. In this study, we examined the combined effect of IL-1 alpha and glucocorticoids on C3 and factor B expression by the endothelial cell. When dexamethasone or hydrocortisone were added to IL-1 alpha, significant potentialization of IL-1 alpha-induced stimulation of C3 and factor B production was observed, occurring at various concentrations of either stimuli. Dose-response experiments indicate that, in vitro, optimal concentrations are in the range of 10(-7) to 10(-5) M for dexamethasone and 50-200 U for IL-1 alpha. In contrast, dexamethasone counteracts, in an additive way, the inhibitory effect of IL-1 alpha on regulatory complement protein factor H production by EC. Such a potentialization between glucocorticoids and IL-1 alpha was not observed for another marker of endothelial activation, IL-1 alpha-induced stimulation of coagulation tissue factor expression. The association of glucocorticoids and IL-1 alpha therefore appears to be a specific and major stimulus for the secretion of complement C3 and factor B, two acute-phase proteins, by the endothelium. As a result of the in vitro endothelium stimulation by glucocorticoids and IL-1 alpha, C3a is generated in the vicinity of the endothelial cell. This study further suggests that complement activation, with its deleterious consequences, may result from the stimulation of endothelium in situations where high levels of IL-1 alpha and endogenous glucocorticoids coexist, such as in septic shock.

Synthesis of the third component of complement (C3) by human gastric cancer-derived cell lines.

This is a study of complement components secreted by gastric cancer-derived cell lines (MKN28, MKN45, MKN74 and KATO-III), each of which has a different histological origin. Haemolytic activity of complement component was detected only in the culture supernatant of KATO-III (C2 activity) and in that of MKN45 (C5 activity). However, the third component of complement, C3, was detected by an ELISA assay in the supernatants of all cell lines. In our studies focusing on C3 production by these cell lines, we have found that: (i) tumour necrosis factor (TNF) induced an increase in the amount of secreted C3 in a dose- and time-dependent fashion; (ii) TNF (10 U/ml) stimulated C3 secretion by these cell lines to levels of 25.4-62.9 ng C3/10(6) cells per 24 hours; (iii) C3 haemolytic activity was detected in supernatants of TNF-stimulated cell lines. The mean specific activities of C3 by TNF (10 U/ml)-stimulated cell lines were 1.2-5.6 x 10(5) effective molecules/ng (e.m./ng), when that of C3 in normal human serum (NHS) was 1.7 x 10(6) e.m./ng; (iv) de novo synthesis of C3 by these cell lines was demonstrated by the effect of cycloheximide and by the incorporation of 35S-methionine into secreted C3; (v) immunoblot analysis of culture supernatants indicated that secreted C3 was mainly composed of C3 alpha and C3 beta chains, but pro-C3 was also present. These results, which show the de novo synthesis and secretion of C3 by all the tested gastric cancer-derived cell lines in response to TNF, suggest the possibility that C3 may be secreted in the gastric wall as part of its normal physiology, or as a result of tumour pathology, and thereby participate in local immune or inflammatory responses.

All-or-None Activation of CRAC Channels by Agonist Elicits Graded Responses in Populations of Mast Cells

In nonexcitable cells, receptor stimulation evokes Ca(2+) release from the endoplasmic reticulum stores followed by Ca(2+) influx through store-operated Ca(2+) channels in the plasma membrane. In mast cells, store-operated entry is mediated via Ca(2+) release-activated Ca(2+) (CRAC) channels. In this study, we find that stimulation of muscarinic receptors in cultured mast cells results in Ca(2+)-dependent activation of protein kinase Calpha and the mitogen activated protein kinases ERK1/2 and this is required for the subsequent stimulation of the enzymes Ca(2+)-dependent phospholipase A(2) and 5-lipoxygenase, generating the intracellular messenger arachidonic acid and the proinflammatory intercellular messenger leukotriene C(4). In cell population studies, ERK activation, arachidonic acid release, and leukotriene C(4) secretion were all graded with stimulus intensity. However, at a single cell level, Ca(2+) influx was related to agonist concentration in an essentially all-or-none manner. This paradox of all-or-none CRAC channel activation in single cells with graded responses in cell populations was resolved by the finding that increasing agonist concentration recruited more mast cells but each cell responded by generating all-or-none Ca(2+) influx. These findings were extended to acutely isolated rat peritoneal mast cells where muscarinic or P2Y receptor stimulation evoked all-or-none activation of Ca(2+)entry but graded responses in cell populations. Our results identify a novel way for grading responses to agonists in immune cells and highlight the importance of CRAC channels as a key pharmacological target to control mast cell activation.

A pure population of lung alveolar epithelial type II cells derived from human embryonic stem cells

Alveolar epithelial type II (ATII) cells are small, cuboidal cells that constitute approximately 60% of the pulmonary alveolar epithelium. These cells are crucial for repair of the injured alveolus by differentiating into alveolar epithelial type I cells. ATII cells derived from human ES (hES) cells are a promising source of cells that could be used therapeutically to treat distal lung diseases. We have developed a reliable transfection and culture procedure, which facilitates, via genetic selection, the differentiation of hES cells into an essentially pure (>99%) population of ATII cells (hES-ATII). Purity, as well as biological features and morphological characteristics of normal ATII cells, was demonstrated for the hES-ATII cells, including lamellar body formation, expression of surfactant proteins A, B, and C, alpha-1-antitrypsin, and the cystic fibrosis transmembrane conductance receptor, as well as the synthesis and secretion of complement proteins C3 and C5. Collectively, these data document the successful generation of a pure population of ATII cells derived from hES cells, providing a practical source of ATII cells to explore in disease models their potential in the regeneration and repair of the injured alveolus and in the therapeutic treatment of genetic diseases affecting the lung.

The complement component C3 plays a critical role in both T H1 and T H2 responses to antigen

Complement component C3 is synthesized by keratinocytes and is activated after skin injury. C3 is also synthesized by peritoneal macrophages, which are activated by the adjuvant alum. We sought to investigate the role of C3 in inciting allergic skin Inflammation and systemic immune responses after epicutaneous sensitization or intraperitoneal sensitization with antigen. C3-deficient (C3-/-) mice and wild-type (WT) control animals were subjected to epicutaneous sensitization with the antigen ovalbumin (OVA) on shaved and tape-stripped skin or intraperitoneal immunization with OVA in alum. Skin Infiltration by eosinophils and expression of mRNA encoding the TH2 cytokines IL-4 and IL-5 in OVA-sensitized skin sites was impaired in C3-/- mice. Splenocytes from epicutaneously sensitized C3-/- mice secreted less IL-4, IL-5, IL-13, and IFN-gamma in response to OVA stimulation than splenocytes from WT control animals. The defect in cytokine secretion by splenocytes was also observed after intraperitoneal immunization of C3-/- mice. C3-/- mice had impaired IgG1, IgG2a, and IgE antibody responses after both epicutaneous and intraperitoneal immunization. The defect in cytokine secretion of C3-/- mice was not due to defective proliferation to antigen, was not observed after anti-CD3 stimulation, and was corrected by the addition of purified C3 protein. These results suggest that C3 plays an important role in both the TH1 and TH2 response to antigen in vivo. The complement pathway might be a potential target in the therapy of allergic diseases.

CD40 Ligand Increases Complement C3 Secretion by Proximal Tubular Epithelial Cells

Interstitial leukocyte infiltration is a major finding in tubulointerstitial damage (TID). Infiltrating lymphocytes interact with proximal tubular epithelial cells (PTEC) by means of secreted soluble factors and/or cell contact mechanisms. CD40 expressed onto PTEC can be engaged by CD40L present on T cells. PTEC are able to locally secrete complement C3, which may most likely promote TID. The aim of the study was to investigate the putative action of CD40 ligation on enhancement of C3 secretion by PTEC. Primary human PTEC and stabilized HK-2 cells were used in culture experiments. Cells were stimulated by soluble factors IL-1beta, IFN-gamma, and/or CD40L-expressing murine fibroblast L cells. Analysis of C3 gene expression was evaluated by reverse-transcription PCR and Northern blot. Secreted C3 was assayed by ELISA and a functional hemolytic test on supernatants. Intracellular events were explored by the NF-kappaB-specific inhibitor caffeic acid phenetyl ester (CAPE). Among soluble factors, IL-1beta and IFN-gamma increased C3 gene expression and secretion (two-fold to three-fold versus basal) on both HK-2 and PTEC. CD40 engagement by CD40L upregulated HK-2 C3 secretion by four-fold. IL-1beta did not further increase CD40-induced C3 secretion, whereas IFN-gamma associated with CD40L was the strongest stimulus (30-fold increase). Inhibition of NF-kappaB offset CD40L-induced C3 secretion by 70%. CD40 ligation is able to enhance C3 secretion by PTEC. This cell contact mechanism is in synergism with a T cell-derived soluble factor (IFN-gamma). C3 secretion induced by CD40L may represent a mechanism of amplification of TID associated with lymphocyte infiltration.

Molecular analysis of a novel hereditary C3 deficiency with systemic lupus erythematosus

A case of inherited homozygous complement C3 deficiency (C3D) in a patient with systemic lupus erythematosus (SLE) and the molecular basis for this deficiency are reported. A 22-year-old Japanese male was diagnosed as having SLE and his medical history revealed recurrent tonsillitis and pneumonia. He was diagnosed as having C3D because of undetectable serum C3 level. His parents were consanguineous. Sequence analysis of C3D cDNA revealed a homozygous deletion of exon 39 (84 bp). A single base substitution (AG to GG) in the 3′-splice acceptor site of intron 38 was identified by sequencing the genomic DNA. Expression of C3Δ(ex39) cDNA, the C3cDNA lacking exon 39, in COS-7 cells revealed that C3Δ(ex39) was retained in endoplasmic reticulum–Golgi intermediate compartment because of defective secretion. These data indicate that a novel AG → GG 3′-splice acceptor site mutation in intron 38 caused aberrant splicing of exon 39, resulting in defective secretion of C3.

Recombinant rat IL-1β and IL-6 synergistically enhance C3 mRNA levels and complement component C3 secretion by H-35 rat hepatoma cells

Hepatic synthesis of complement component C3 is regulated in part by inflammatory cytokines. Rat models are frequently employed to investigate pathogenic roles of complement and cytokines. However, cytokines obtained from species other than the rat were used in previous studies of cytokine regulation of C3 synthesis in rat hepatocytes or hepatoma cells. It is not known whether these prior reports predict hepatocellular responses evoked by rat cytokines. Therefore, H-35 rat hepatoma cells were employed to measure the effect of recombinant rat IL-1β, IL-6, IFN-γ, and TNF-α on C3 protein secretion and C3 mRNA levels quantified by ELISA and quantitative RT-PCR. Compared to untreated control cells, H-35 cells treated with IL-1β, IL-6, and IFN-γ increased C3 secretion approximately 10-, 4-, and 2-fold, respectively. TNF-α was toxic, precluding further analysis. IL-1β and IL-6 demonstrated synergy with respect to the quantity and rate of increase of C3 mRNA measured and the magnitude of C3 protein secretion. Previous reports using non-rat cytokines did not consistently predict H-35 responses to rat cytokines. Consequently, we recommend the use of rat cytokines in rat models that include analysis of cytokine-mediated events.

Exposure to Organic Dust Causes Activation of Human Plasma Complement Factors C3 and B and the Synthesis of Factor C3 by Lung Epithelial Cells In Vitro

Exposure in swine confinement buildings induces an intense airway inflammation. Twenty-two volunteers, of whom eleven wore a half-mask, were exposed for 3 hr in a swine barn. Blood samples were drawn before and after exposure. The ratio C3b/totalC3 in plasma decreased from 6.8 to 5.0% (p = 0.02) without mask and from 6.6 to 5.9% (p = 0.01) with mask (p = 0.67 between groups). The ratio Bb/totalB decreased from 14.5 to 13.5% (p < 0.01) without and 14.6-13.3% (p = 0.09) with mask (p = 0.25 between groups). Epithelial cells (A549) incubated up to 24 hr with 0.1 mg/mL dust suspensions were analysed for C3, IL-6 and IL-8 secretion. Cumulative C3 synthesis of dust stimulated cell cultures was 43,000 pg/mL compared to 25,000 pg/mL in unstimulated cells. Cumulative dust-induced IL-6 and IL-8 secretion was 200 and 3000 pg/mL, respectively and below detection in unstimulated cells. The activation of complement in vivo and induced C3 synthesis by epithelial cells suggests a role of complement in the airway reaction to organic dust exposure.

Close Functional Coupling between Ca2+ Release-activated Ca2+ Channels, Arachidonic Acid Release, and Leukotriene C4 Secretion

In non-excitable cells, one major route for Ca2+ influx is through store-operated Ca2+ channels in the plasma membrane. These channels are activated by the emptying of intracellular Ca2+ stores, and in some cell types, particularly of hemopoietic origin, store-operated influx occurs through Ca2+ release-activated Ca2+ (CRAC) channels. However, little is known about the downstream consequences of CRAC channel activation. Here, we report that Ca2+ entry through CRAC channels stimulates arachidonic acid production, whereas Ca2+ release from the stores is ineffective even though the latter evokes a robust intracellular Ca2+ signal. We find that arachidonic acid released by Ca2+ entering through CRAC channels is used to synthesize the potent paracrine proinflammatory signal leukotriene C4 (LTC4). Both pharmacological inhibitors of CRAC channels and mitochondrial depolarization, which impairs CRAC channel activity, suppress arachidonic acid release and LTC4 secretion. Thus, arachidonic acid release is preferentially stimulated by elevated subplasmalemmal Ca2+ levels due to open CRAC channels, suggesting that the enzyme is located close to the CRAC channels. Our results also identify a novel role for CRAC channels, namely the activation of a downstream signal transduction pathway resulting in the secretion of LTC4. Finally, mitochondria are key determinants of the generation of both intracellular (arachidonic acid) and paracrine (LTC4) signals through their effects on CRAC channel activity.