A C3(H20) recycling pathway is a component of the intracellular complement system

An intracellular complement system (ICS) has recently been described in immune and nonimmune human cells. This system can be activated in a convertase-independent manner from intracellular stores of the complement component C3. The source of these stores has not been rigorously investigated. In the present study, Western blotting identified a band corresponding to C3 in freshly isolated human peripheral blood cells that was absent in corresponding cell lines. One difference between native cells and cell lines was the time absent from a fluid-phase complement source; therefore, we hypothesized that loading C3 from plasma was a route of establishing intracellular C3 stores. We found that many types of human cells specifically internalized C3(H(2)O), the hydrolytic product of C3, and not native C3, from the extracellular milieu. Uptake was rapid, saturable, and sensitive to competition with unlabeled C3(H(2)O), indicating a specific mechanism of loading. Under steady-state conditions, approximately 80% of incorporated C3(H(2)O) was returned to the extracellular space. These studies identify an ICS recycling pathway for C3(H(2)O). The loaded C3(H(2)O) represents a source of C3a, and its uptake altered the cytokine profile of activated CD4(+) T cells. Importantly, these results indicate that the impact of soluble plasma factors should be considered when performing in vitro studies assessing cellular immune function

Complement C3 serum levels in anorexia nervosa: a potential biomarker for the severity of disease?

BackgroundAnorexia nervosa carries the highest mortality rate of any psychiatric disorder. Even the most critically ill anorexic patients may present with normal 'standard' laboratory values, underscoring the need for a new sensitive biomarker. The complement cascade, a major component of innate immunity, represents a driving force in the pathophysiology of multiple inflammatory disorders. The role of complement in anorexia nervosa remains poorly understood. The present study was designed to evaluate the role of complement C3 levels, the extent of complement activation and of complement hemolytic activity in serum, as potential new biomarkers for the severity of anorexia nervosa.Patients and methodsThis was a prospective cohort study on 14 patients with severe anorexia nervosa, as defined by a body mass index (BMI) <14 kg/m2. Serum samples were obtained in a biweekly manner until hospital discharge. A total of 17 healthy subjects with normal BMI values served as controls. The serum levels of complement C3, C3a, C5a, sC5b-9, and of the 50% hemolytic complement activity (CH50) were quantified and correlated with the BMIs of patients and control subjects.ResultsSerum C3 levels were significantly lower in patients with anorexia nervosa than in controls (median 3.7 (interquartile range (IQR) 2.5-4.9) vs 11.4 (IQR 8.9-13.7, P <0.001). In contrast, complement activation fragments and CH50 levels were not significantly different between the two groups. There was a strong correlation between index C3 levels and BMI (Spearman correlation coefficient = 0.71, P <0.001).ConclusionsComplement C3 serum levels may represent a sensitive new biomarker for monitoring the severity of disease in anorexia nervosa. The finding from this preliminary pilot study will require further investigation in future prospective large-scale multicenter trials

The versatile functions of complement C3-derived ligands

The complement system is a major component of immune defense. Activation of the complement cascade by foreign substances and altered self-structures may lead to the elimination of the activating agent, and during the enzymatic cascade, several biologically active fragments are generated. Most immune regulatory effects of complement are mediated by the activation products of C3, the central component. The indispensable role of C3 in opsonic phagocytosis as well as in the regulation of humoral immune response is known for long, while the involvement of complement in T-cell biology have been revealed in the past few years. In this review, we discuss the immune modulatory functions of C3-derived fragments focusing on their role in processes which have not been summarized so far. The importance of locally synthesized complement will receive special emphasis, as several immunological processes take place in tissues, where hepatocyte-derived complement components might not be available at high concentrations. We also aim to call the attention to important differences between human and mouse systems regarding C3-mediated processes. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Lt

Renal C3 complement component: feed forward to diabetic kidney disease

BACKGROUND: Diabetic nephropathy is the main cause of end-stage renal disease and has reached epidemic proportions. METHODS: Comprehensive genomic profiling (RNAseq) was employed in the ZS (F1 hybrids of Zucker and spontaneously hypertensive heart failure) model of diabetic nephropathy. Controls were lean littermates. RESULTS: Diabetic nephropathy in obese, diabetic ZS was accelerated by a single episode of renal ischemia (DI). This rapid renal decline was accompanied by the activation of the renal complement system in DI, and to a lesser extent in sham-operated diabetic rats (DS). In DI there were significant increases in renal mRNA encoding C3, C4, C5, C6, C8, and C9 over sham-operated lean normal controls (LS). Moreover, mRNAs encoding the receptors for the anaphylatoxins C3a and C5a were also significantly increased in DI compared to LS. The classic complement pathway was activated in diabetic kidneys with significant increases of C1qa, C1qb, and C1qc mRNAs in DI over LS. In addition, critical regulators of complement activation were significantly attenuated in DI and DS. These included mRNAs encoding CD55, decay accelerating factor, and CD59, which inhibit the membrane attack complex. C3, C4, and C9 proteins were demonstrated in renal tubules and glomeruli. The complement RNAseq data were incorporated into a gene network showing interactions among C3-generating renal tubular cells and other immune competent migratory cells. CONCLUSIONS: We conclude that local activation of the complement system mediates renal injury in diabetic nephropathy

Synthesis and propagation of complement C3 by microglia/monocytes in the aging retina

INTRODUCTION Complement activation is thought to contribute to the pathogenesis of age-related macular degeneration (AMD), which may be mediated in part by para-inflammatory processes. We aimed to investigate the expression and localization of C3, a crucial component of the complement system, in the retina during the course of aging. METHODS SD rats were born and reared in low-light conditions, and euthanized at post-natal (P) days 100, 450, or 750. Expression of C3, IBA1, and Ccl- and Cxcl- chemokines was assessed by qPCR, and in situ hybridization. Thickness of the ONL was assessed in retinal sections as a measure of photoreceptor loss, and counts were made of C3-expressing monocytes. RESULTS C3 expression increased significantly at P750, and correlated with thinning of the ONL, at P750, and up-regulation of GFAP. In situ hybridization showed that C3 was expressed by microglia/monocytes, mainly from within the retinal vasculature, and occasionally the ONL. The number of C3-expressing microglia increased significantly by P750, and coincided spatiotemporally with thinning of the ONL, and up-regulation of Ccl- and Cxcl- chemokines. CONCLUSIONS Our data suggest that recruited microglia/monocytes contribute to activation of complement in the aging retina, through local expression of C3 mRNA. C3 expression coincides with age-related thinning of the ONL at P750, although it is unclear whether the C3-expressing monocytes are a cause or consequence. These findings provide evidence of activation of complement during natural aging, and may have relevance to cellular events underling the pathogenesis of age-related retinal diseases.Funding provided by Australian Research Council Centres of Excellence Program Grant (CE0561903)

Complement Component C3 and Complement Factor B Promote Growth of Cutaneous Squamous Cell Carcinoma

Cutaneous squamous cell carcinoma (cSCC) is one of the most common metastatic skin cancers with increasing incidence. We examined the roles of complement component C3 and complement factor B (CFB) in the growth of cSCC. Analysis of cSCC cell lines (n = 8) and normal human epidermal kerati-nocytes (n = 11) with real-time quantitative PCR and Western blotting revealed up-regulation of C3 and CFB expression in cSCC cells. Immunohistochemical staining revealed stronger tumor cell specific Labeling for C3 and CFB in invasive cSCCs (n = 71) and recessive dystrophic epidermolysis bullosa-associated cSCCs (n = 11) than in cSCC in situ (n = 69), actinic keratoses (n = 63), and normal skin (n = 5). Significant up-regulation of C3 and CFB mRNA expression was noted in chemically induced mouse cSCCs, compared to benign papillomas. Knockdown of C3 and CFB expression inhibited migration and proliferation of cSCC cells and resulted in potent inhibition of extracellular signal regulated kinase 1/2 activation. Knockdown of C3 and CFB markedly inhibited growth of human cSCC xenograft tumors in vivo. These results provide evidence for the rotes of C3 and CFB in the development of cSCC and identify them as biomarkers and potential therapeutic targets in this metastatic skin cancer.Peer reviewe

Pharmacological management of immune and oxidative disturbances in patients with encephalopathy on the background of hypertension

Inclusion of a comprehensive drug treatment of patients with discirculatory encephalopathy cerebrolysin combination with meksidol normalizes the concentration of C3 and C4 components of the complement system, IgG, malondialdehyde, catalase activity, total antioxidant activity of blood serum, corrects, but not to the level of standards, the contents of cytokines (TNF, IL-1β, IL-6, IL-8, IL-18, IFγ, IL-2, IL-17), complement component C5, IgA, acylhydrohyperoxide, neopterin and increases anti-inflammatory cytokine

Immunology: Investigations on the cell type responsible for the endometrial secretion of complement component 3 (C3)

It has been shown that and human endometria have the capacity to produce complement component 3 (C3). In rats, endometrial C3 is an oestrogen-dependent protein produced and secreted by glandular cells. The cell responsible for the synthesis and secretion of human endometrial C3 has not been clearly defined. Our study was aimed at answering this question. Samples of endometrium obtained from hysterectomies were either immunostained for C3 or digested with collagenase; then the stromal and glandular cells were separated and immunopurified (or not) with an antibody to CD45 coupled to magnetic beads to eliminate the endometrial lymphomyeloid cells. Cells were cultured for 2 weeks and C3 measured in the medium by an in-house radioimmunoassay. Glandular as well as stromal cells stained positively for C3 and released C3 in vitro. The release of C3 from both cell types could be inhibited by cycloheximide. Epithelial cells produced significantly more C3 than stromal cells, and endometrial C3 production was higher for both cell types when these were obtained from secretory as compared to proliferative endometria. Lymphomyeloid cells were possibly a source of C3 since after immunoadsorption of these cells, the remaining stromal or glandular cells produced significantly less C3. We conclude that endometrial stromal, glandular and lymphomyeloid cells all produce C

A new role for complement C3: regulation of antigen processing through an inhibitory activity.

International audienceIncreasing evidence underlines the involvement of complement component C3 in the establishment of acquired immunity which appears to play a complex role and to act at different levels. As antigen proteolysis by antigen presenting cells is a key event in the control of antigen presentation efficiency, and consequently in the quality of the immune response, we investigated whether C3 could modulate this step. Our results demonstrate for the first time that C3 can interfere with antigen proteolysis: (i) proteolysis of tetanus toxin (TT) by the lysosomal fraction from a human monocytic cell line (U937) is impaired in the presence of C3, (ii) this effect is C3-specific and involves the C3c fragment of the protein, (iii) C3c is effective even after disulfide disruption, but none of its three constitutive peptides is individually accountable for this inhibitory effect and (iv) the target-protease(s) exhibit(s) a serine-protease activity. The physiological relevance of our results is demonstrated by experiments showing a subcellular colocalisation of TT and C3 after their uptake by U937 and the reduction of TT proteolysis once internalised together with C3. These results highlight a novel role for C3 that broadens its capacity to modulate acquired immune response

Complement system and alcoholic liver disease

Alcoholic liver disease (ALD) is a well recognized and growing health problem worldwide. ALD advances from fatty liver to inflammation, necrosis, fibrosis and cirrhosis. There is accumulating evidence that the innate immune system is involved in alcoholic liver injury. Within the innate and acquired immune systems, the complement system participates in inflammatory reactions and in the elimination of invading foreign, as well as endogenous apoptotic or injured cells. The present study aimed at evaluating the role of the complement system in the development of alcoholic liver injury. First, in order to study the effects of chronic ethanol intake on the complement system, the deposition of complement components in liver and the expression of liver genes associated with complement in animals with alcohol-induced liver injury were examined. It was demonstrated that chronic alcohol exposure leads to hepatic deposition of the complement components C1, C3, C8 and C9 in the livers of rats. Liver gene expression analysis showed that ethanol up-regulated the expression of transcripts for complement factors B, C1qA, C2, C3 and clusterin. In contrast, ethanol down-regulated the expression of the complement regulators factor H, C4bp and factor D and the terminal complement components C6, C8α and C9. Secondly, the role of the terminal complement pathway in the development of ALD was evaluated by using rats genetically deficient in the complement component C6 (C6-/-). It was found that chronic ethanol feeding induced more liver pathology (steatosis and inflammatory changes) in C6-/- rats than in wild type rats. The hepatic triacylglyceride content and plasma alanine aminotransferase activity increased in C6-/- rats, supporting the histopathological findings and elevation of the plasma pro-/anti-inflammatory TNF-/IL-10 ratio was also more marked in C6-/- rats. Third, the role of the alternative pathway in the development of alcoholic liver steatosis was characterized by using C3-/- mice. In C3-/- mice ethanol feeding tended to reduce steatosis and had no further effect on liver triacylglyceride, liver/body weight ratio nor on liver malondialdehyde level and serum alanine aminotransferase activity. In C3-/- mice alcohol-induced liver steatosis was reduced also after an acute alcohol challenge. In both wild type and C3-/- mice ethanol markedly reduced serum cholesterol and ApoA-I levels, phospholipid transfer protein activity and hepatic mRNA levels of fatty acid binding proteins and fatty acid -oxidation enzymes. In contrast, exclusively in C3-/- mice, ethanol treatment increased serum and liver adiponectin levels but down-regulated the expression of transcripts of lipogenic enzymes, adiponectin receptor 2 and adipose differentiation-related protein and up-regulated phospholipase D1. In conclusion, this study has demonstrated that the complement system is involved in the development of alcohol-induced liver injury. Chronic alcohol exposure causes local complement activation and induction of mRNA expression of classical and alternative pathway components in the liver. In contrast expression of the terminal pathway components and soluble regulators were decreased. A deficient terminal complement pathway predisposes to alcoholic liver damage and promotes a pro-inflammatory cytokine response. Complement component C3 contributes to the development of alcohol-induced fatty liver and its consequences by affecting regulatory and specific transcription factors of lipid homeostasis.Alkoholin aiheuttamat sairaudet ovat tämänhetkisen terveydenhuollon keskeisimpiä ongelmia ja voimakkaasti lisääntymässä sekä Suomessa että muualla maailmassa. Alkoholimaksavaurio alkaa maksan rasvoittumisesta ja etenee maksakirroosiin. Rasvamaksa voi kehittyä myös ilman alkoholia ylipainoisilla, joiden osuus väestössä on kasvussa. Maksa rasvoittuu jo yhden viikonlopun rajun ryypiskelyn seurauksena, mutta tila on palautuva jollei alkoholin käyttöä jatketa. Maksakirroosi sen sijaan on palautumaton vaurio, johon kuitenkin vain 15-25% alkoholisteista sairastuu. Alkoholin lisäksi täytyy siis olla muita yksilöllisiä tekijöitä, jotka laukaisevat vaurion kehittymisen. Suurin osa näistä tekijöistä on tuntemattomia. Tämän tutkimuksen lähtökohtana oli oletus, että kehon puolustus- ja puhtaanpitojärjestelmien tehokkuus vaikuttaa alttiuteen saada alkoholin aiheuttama maksasairaus. Kehon komplementtijärjestelmä on keskeinen immuunijärjestelmän osa. Komplementin tehtäviin kuuluu yhdessä tulehdussolujen kanssa poistaa kehosta haitallisia rakenteita, kuten mikrobeja ja vaurioituneita kudosrakenteita, sekä aikaansaada tulehdusreaktio. Tutkimustyössä tutkittiin synnynnäisten komplementtipuutosten vaikutusta alkoholimaksavaurion kehittymiseen eläinmalleissa. Rotille, joilla oli komplementtitekijän C6 puutos, kehittyi vaikeampi maksasairaus ja enemmän tulehdusmuutoksia kuin kontrollirotille kuuden viikon alkoholialtistuksen ja rasvapitoisen ruoan jälkeen. Tämä viittaa siihen, että komplementin lopputien toiminta suojaa maksaa alkoholin aiheuttamilta kudosvaurioilta. Tämä voi liittyä siihen, että komplementin solutuhokompleksi mahdollisesti estää alkoholin vaurioittamasta suolesta päässeitä mikrobeja aiheuttamasta kudostuhoa. Komplementtijärjestelmällä ja rasva-ainemetabolialla on havaittu olevan yhteyksiä keskenään. Alkoholi aiheuttaa rasvapitoista ruokaa syöville hiirille rasvamaksavaurion ja sitä seuraavan tulehdusreaktion. Mikäli hiiriltä puuttui komplementin tekijä C3 ei maksaan kertynyt rasvaa eikä tulehdusmuutoksiakaan kehittynyt. Tämä merkittävä tulos osoittaa, että komplementti ja erityisesti sen tekijä C3 osallistuu alkoholin aiheuttaman tulehduksellisen maksavaurion kehittymiseen. C3-puutoksen vaikutus on näin eri suuntainen kuin tekijän C6 puutos. C3 tekijä ja sen aktivaatiotuotteet vaikuttavat rasvametaboliaan lisäten triglyseridien kertymistä kudoksiin ja aiheuttaen tulehdusreaktioita. Hiirimallitutkimus osoitti selvästi ja ensimmäistä kertaa maailmassa, että C3 on olennainen tekijä alkoholivälitteisen maksavaurion kehittymisessä. C3-puutoksen vaikutusta maksavaurioon ja rasva-aineenvaihduntaan tutkittiin laajemmin selvittämällä alkoholin aiheuttamia geeniluennan muutoksia C3-puutteisilla ja normaaleilla hiirillä. C3 tekijän todettiin lisäävän hiirillä rasva-aineenvaihdunnan entsyymien ja adiponektiinireseptori 2:n määriä maksassa, mutta vähentävän suojaavan adiponektiinin määrää sekä maksassa että seerumissa. Komplementtijärjestelmällä ja rasva-aineenvaihdunnalla on näin ollen merkittäviä vuorovaikutuksia keskenään. Etanoli vaikutti suoraan myös komplementtijärjestelmän toimintaan. Se lisäsi komplementin aktivaatioon tarvittavien klassisen ja oikotin aktivaatioreittien tekijöiden geeniluentaa, mutta vähensi säätelijämolekyylien ja loppupään tekijöiden geenien luentaa. Yhteisvaikutus on näin ollen tulehdusta voimistava. Geenianalyyseissä voitiin myös todeta etanolin lisäävän kahden proteiinin, SPARCin ja lipokaliini-2:n tuottoa. Tämä antaa aiheen tutkia näiden molekyylien käyttöä varhaisina merkkiaineina alkoholin aiheuttaman maksavaurion toteamisessa. Tehdyt tutkimukset ovat paljastaneet komplementtijärjestelmän osallistuvan keskeisesti alkoholimaksavaurion kehittymiseen. Muita päätuloksia ovat yhteyden toteaminen rasvametabolian ja komplementin välillä sekä mahdollisten uusien merkkiaineiden löytäminen alkoholivälitteisen maksavaurion toteamiseen