Fifth Component Of Complement Research Articles

Antioxidant and C5a-blocking strategy for hepatic ischemia\u2013reperfusion injury repair

BackgroundNonspecific liver uptake of nanomaterials after intravenous injection has hindered nanomedicine for clinical translation. However, nanomaterials’ propensity for liver distribution might enable their use in hepatic ischemia–reperfusion injury (IRI) repair. During hepatic IRI, reactive oxygen species (ROS) are generated and the fifth component of complement (C5a) is activated. In addition, C5a is confirmed to exacerbate the vicious cycle of oxidative stress and inflammatory damage. For these reasons, we have investigated the development of nanomaterials with liver uptake to scavenge ROS and block C5a for hepatic IRI repair.ResultsTo achieve this goal, a traditional nanoantioxidant of nanoceria was surface conjugated with the anti-C5a aptamers (Ceria@Apt) to scavenge the ROS and reduce C5a-mediated inflammation. High uptake of Ceria@Apt in the liver was confirmed by preclinical positron emission tomography (PET) imaging. The clinical symptoms of hepatic IRI were effectively alleviated by Ceria@Apt with ROS scavenging and C5a blocking in mice model. The released pro-inflammatory cytokines were significantly reduced, and subsequent inflammatory reaction involved in the liver was inhibited.ConclusionsThe synthesized Ceria@Apt has great potential of medical application in hepatic IRI repair, which could also be applied for other ischemic-related diseases.Graphic abstract

A simple PCR-based method for the rapid genotyping of inherited fifth complement component (C5)-deficient mice.

The fifth component of complement (C5) is considered to be the center of complement activation and function. However, there are no genetically engineered knockout mice for this gene, and the only commercially available inherited C5-deficient mice, in which a “TA” nucleotide deletion in the coding frame was previously identified, are in theC57BL/10Sn genetic background rather than the commonly used backgrounds C57BL/6 and BALB/c. Therefore, these mice must be backcrossed into the desired genetic background. Here, we developed an ARMS (amplification refractory mutation system) PCR method using a specific primer pair that was able to discriminate between the genotypes when the resulting product was analyzed by agarose gel electrophoresis. These results were supported by quantitative RT-PCR and semi-quantitative PCR and were consistent with the results from sequencing each backcrossed generation. Using ARMS-PCR method, we generated C5-deficient mice in the C57BL/6 background over 9 backcrossed generations and further verified the phenotype using complement-mediated hemolytic assays. In this study, we describe a simple, rapid and reliable PCR-based method for genotyping inherited C5-deficient mice that may be used to backcross C57BL/10Sn mice into other genetic backgrounds.

Deficiency of the fifth component of complement in a young South African woman; three putative genetic defects

Deficiency of the fifth component of complement in a young South African woman; three putative genetic defects

Variability of biological effects of silicas: Different degrees of activation of the fifth component of complement by amorphous silicas

A biogenic and a pyrogenic amorphous silica were incubated in normal human plasma and compared on a per unit surface basis for their ability to split C5 molecules and yield small C5a peptides. Since C5a peptides induce selective chemotactic attraction of polymorphonuclear leukocytes (PMN), measurement of PMN-induced chemotaxis was used as an index of C5 activation. Though to a lesser extent than the crystalline forms, amorphous silicas can promote the cleavage of C5 protein and generation of C5a-like fragment. The biogenic silica, which differs from the pyrogenic variety in particle shape, level of contaminants, and degree of surface hydrophilicity, besides specific surface, induced a greater response. Both silicas activated C5 through a process which seems to involve multiple events similar to those induced by crystalline silica. C5 molecules are adsorbed and hydroxyl radicals are generated through Haber Weiss cycles catalyzed by the redox-active iron present at the particle surface either as trace impurities or chelated from plasma by silanol groups. In turn, these radicals convert native C5 to an oxidized C5-like form C5(H 2O 2). Finally, C5(H 2O 2) is cleaved by protease enzymatic action of plasma kallikrein activated by the same silica dusts, yielding a product, C5a(H 2O 2), having the same functional characteristic as C5a.

Expression and induction of anaphylatoxin C5a receptors in the rat liver.

The C5a-anaphylatoxin which is generated by limited proteolysis upon activation of the fifth component of complement may be induced by the classical, the alternative or the lectin pathway. C5a has been shown, under normal conditions, to induce the release of prostanoids from Kupffer cells (KC) and hepatic stellate cells (HSC) and thereby indirectly to increase glucose output from hepatocytes (HC). A direct action of C5a on HC would require the expression of the specific C5a receptor (C5aR). In studies using quantitative RT-PCR it was shown that non-stimulated HC lack C5aR, in contrast to KC, HSC and sinusoidal endothelial cells (SEC) all of which contained mRNA for the C5aR in decreasing amounts. FACS analyses, immunohisto- and immunocytochemistry as well as functional analyses confirmed the results of the RT-PCR assays. Under inflammatory situations the C5aR was found to be upregulated in various organs and tissues which included the liver. Interleukin-6 (IL-6) as a main inflammatory mediator in the liver induced a de novo expression of functional C5aR in HC in-vitro and in-vivo. In contrast, LPS failed to induce C5aR directly in cultured HC in-vitro but induced C5aR in HC in vivo and in co-cultures of HC and KC which release IL-6 upon stimulation with LPS. So far, the only known effector function of C5a on HSC was the induction of prostanoid release. In an approach to reveal new functions of C5aR in HSC, the cells responsible for liver fibrosis, it could be shown that C5a upregulated fibronectin-specific mRNA five-fold whereas entactin, collagen IV and the structure protein smooth muscle actin were not affected. In addition, C5a did not upregulate specific mRNA for the profibrotic cytokine TGF-beta1 in either isolated KC or HSC. Thus, C5a alone appears to have only a limited role in the induction of liver fibrosis.

Cloning and purification of the rainbow trout fifth component of complement (C5)

To gain further insight into the evolutionary history of the complement proteins C3, C4, and C5 we have now cloned the fifth component of complement from a rainbow trout ( Oncorhynchus mykiss) liver cDNA library; this is the first report of C5 cloning in a species other than human and mouse. The deduced amino acid sequence of a partial cDNA clone (2.25 kb), representing approximately 44% of the coding sequence, showed 60 and 58% similarity to human and mouse C5, respectively. To validate the molecular information derived from the cloning we developed an improved purification protocol. Mass spectrometric analysis of C5 tryptic digests yielded peptide signals that matched theoretical protein sequence derived from the partial cDNA. Northern blot analysis of RNA from various tissues showed the presence of a single mRNA transcript in trout liver and Southern blot analysis indicated that the gene coding for C5 is present as a single copy in the trout genome. The presence of C5 in trout suggests that C3, C4, and C5 must have diverged before the appearance of teleost fish.

Cloning and purification of the rainbow trout fifth component of complement (C5)

Cloning and purification of the rainbow trout fifth component of complement (C5)

B cells are essential for vaccination-induced resistance to virulent Toxoplasma gondii.

T lymphocytes and gamma interferon (IFN-gamma) are known mediators of immune resistance to Toxoplasma gondii infection, but whether B cells also play an important role is not clear. We have investigated this issue using B-cell-deficient (muMT) mice. If vaccinated with attenuated T. gondii tachyzoites, muMT mice are susceptible to a challenge intraperitoneal infection with highly virulent tachyzoites that similarly vaccinated B-cell-sufficient mice resist. Susceptibility is evidenced by increased numbers of parasites at the challenge infection site and by extensive mortality. The susceptibility of B-cell-deficient mice does not appear to be caused by deficient T-cell functions or diminished capacity of vaccinated and challenged B-cell-deficient mice to produce IFN-gamma. Administration of Toxoplasma-immune serum, but not nonimmune serum, to vaccinated B-cell-deficient mice significantly prolongs their survival after challenge with virulent tachyzoites. Vaccinated mice lacking Fc receptors or the fifth component of complement resist a challenge infection, suggesting that neither Fc-receptor-dependent phagocytosis of antibody-coated tachyzoites nor antibody-dependent cellular cytotoxicity nor antibody-and-complement-dependent lysis of tachyzoites is a crucial mechanism of resistance. However, Toxoplasma-immune serum effectively inhibits the infection of host cells by tachyzoites in vitro. Together, the results support the hypothesis that B cells are required for vaccination-induced resistance to virulent tachyzoites in order to produce antibodies and that antibodies may function protectively in vivo by blocking infection of host cells by tachyzoites.

Cleavage of the Fifth Component of Complement and Generation of a Functionally Active C5b6-like Complex by Human Leukocyte Elastase

Activation by complement C3/CS convertases of the fifth component of human complement, CS, leads to two active cleavage products: CSa, a chemotactic peptide, and CSb, the activated form of CS. Human leukocyte elastase (HLE) has long been known to also release from CS a chemotactic, CSa-like fragment. This, however, cannot be identical with CSa, since HLE does not cleave peptide bonds at the carboxyl group of arginine, the cleavage site that separates CSa and CSb after the exposure to the complement convertases. Therefore, the question arose whether HLE is capable of releasing a functionally CSb-like product from CS. The results show that this is, indeed, so. Treatment of human CS with HLE in the presence of C6 leads to the formation on an active C5b6-like complex that lyses non-sensitized guinea pig red cells upon addition of the terminal components C7, CS, and C9. However, since C6 is highly sensitive to the hydrolytic action of HLE, the yield of the activation complex is rather low. The results offer a third possibility for the activation of CS: 1) classical cleavage at Arg 74 by complement convertases, 2) oxidation of methionine residues without cleavage, and, as shown here, 3) cleavage by elastase at (a) site(s) distal from Arg 74 The three procedures may modulate the relative yield of the two activities generated from CS, CSa-like and CSb-like effects.

Budesonide down-regulates eosinophil locomotion but has no effects on ECP release or on H2O2 production.

Treatment of allergic asthma with inhaled corticosteroids results in local down-regulation of proinflammatory cytokine synthesis and in marked decrease in tissue eosinophilia. Blood concentrations of inhaled corticosteroids, although significantly lower than those measured in the lung, may still have antiinflammatory effects on circulating eosinophils, reducing their ability to migrate. The aim of our study was to evaluate in vitro the activity of budesonide on blood eosinophils by measuring their chemotactic response, eosinophil cationic protein (ECP) release, and hydrogen peroxide (H2O2) production in the presence of different drug concentrations similar to those obtained at airway level (10(-8) and 10(-7) M) and at blood level (10(-10) and 10(-9) M). Partially purified blood eosinophils, isolated from 23 asthmatic subjects, were used to evaluate the activity of budesonide on: (1) chemotaxis toward the activated fifth component of complement (C5a, 0.1 microg/ml) or recombinant human (rh) interleukin (IL)-5 (200 pg/ml), (2) ECP release by cells stimulated with tetradecanoylphorbol acetate (TPA) and (3) H2O2 production by TPA-activated cells. The chemotactic response to C5a was down-regulated significantly by budesonide only by the highest concentrations tested (10(-8) and 10(-7) M); differently, budesonide was effective in inhibiting eosinophil migration toward rhIL-5, at all concentrations tested (p < 0.01, each comparison). By contrast, no drug-induced modifications were observed in ECP release or in H2O2 production (p > 0.05, each comparison). We conclude that concentrations of budesonide similar to those obtained in vivo are effective in inhibiting eosinophil locomotion but not in down-regulating the release of reactive oxygen species and granule-associated proteins.

Modulation of Formyl Peptide Receptor Expression by IL-10 in Human Monocytes and Neutrophils

IL-10, originally described as a cytokine synthesis inhibitory factor, is secreted by a number of cells of the immune system, including monocytes and T cells. Although IL-10 is being assigned as an immunosuppressive cytokine, our study showed that FMLP-R mRNA was rapidly up-regulated by exposure of monocytes to graded concentrations of this cytokine, with maximal (three- to fourfold) stimulation with 10 ng/ml. The effect was rapid, being observable as early as 1 h of treatment with IL-10, maximal between 2 and 4 h, and still evident after 24 h and was associated with an increase of receptor expression on the cell surface as assessed by flow cytometry analysis. Pretreatment of monocytes with actinomycin D completely abrogated the effect of IL-10, suggesting a transcriptional regulation. Moreover, IL-10-treated monocytes showed a significantly enhanced functional responsiveness to FMLP with enhanced (three- to fourfold) chemotaxis and augmented (twofold) intracellular calcium mobilization. In polymorphonuclear neutrophils (PMN), IL-10 also mediated a twofold augmentation of FMLP-R expression. In parallel experiments, we observed that IL-10 could differentially modulate other chemotactic receptors. Hence, we observed that IL-10 augmented two-to threefold platelet-activating factor receptor (PAF-R) expression, whereas it had no significant effect on the fifth component of complement (C5a) receptor (C5a-R) expression. Collectively, our results demonstrate that IL-10 may play an important role in inflammatory process through modulation of chemotactic receptor expression.

DNA vaccination: transfection and activation of dendritic cells as key events for immunity.

The mechanisms underlying initiation and maintenance of CD4 T cell responses after DNA vaccination were studied using a construct coding for nonsecreted fifth component of complement (C5) protein, thus restricting the availability of antigen. The only cell types to express C5 were keratinocytes at the site of DNA application and a small number of dendritic cells present in the draining lymph nodes. Antigen expression persisted for up to 12 wk in keratinocytes, but dendritic cells did not express C5 beyond 2 wk after vaccination. Cross-priming of dendritic cells by C5 expressed in keratinocytes did not occur unless keratinocyte death was induced by irradiation in vitro. CD4 T cells were activated in the draining lymph nodes only and subsequently migrated to the spleen, where memory T cells persisted for longer than 40 wk despite the absence of a source of persistent antigen. While DNA vaccination resulted in transfection of a small proportion of dendritic cells only, it led to general activation of all dendritic cells, thus providing optimal conditions for effective T cell activation and maintenance of memory.

Normal Thymic Selection of TCR Transgenic CD4 T Cells, but Impaired Survival in the Periphery Despite the Presence of Selecting MHC Molecules

In this paper, we investigate selection in the thymus and survival in the periphery of CD4 T cells, which carry a major histocompatibility class II-restricted transgenic TCR (A18 TCRtg) specific for a natural self Ag, the fifth component of complement (C5). A18 TCRtg thymocytes develop normal numbers of CD4 single-positive (SP) thymocytes, but do not show pronounced overselection as do some other TCR transgenic strains. CD4 SP cells are mature as judged by termination of CD8 synthesis, resistance to cortisone, and functional competence. The kinetics of positive selection, determined by BrdU labeling, are very fast. CD4 SP thymocytes are demonstrable within 2 days of labeling, and within 8 days after labeling a large proportion (20%) of lymph node T cells are recent thymic emigrants. The high number of recent thymic emigrants suggests rapid turnover of CD4 T cells in the periphery, which was confirmed by thymectomy and determination of CD4 T cell life spans. A18 TCRtg T cells have a t(1/2) of approximately 6 wk, despite the presence of selecting MHC molecules. This explains the failure to accumulate high numbers of peripheral T cells and suggests that the MHC-bound ligand(s) responsible for initiating survival signals is limiting for the selection and maintenance of A18 transgenic CD4 T cells.

Multiple Signaling Pathways of Human Interleukin-8 Receptor A: INDEPENDENT REGULATION BY PHOSPHORYLATION

Interleukin-8 (IL-8) receptor A (CXCR1) couples to a pertussis toxin-sensitive G protein to mediate phospholipase Cbeta (PLCbeta) activation and cellular responses. Responses to CXCR1 are attenuated by prior exposure of neutrophils to either IL-8, a cleavage product of the fifth component of complement (C5a) or n-formylated peptides (formylmethionylleucylphenylalanine, fMLP). To characterize the role of receptor phosphorylation in the regulation of the CXCR1, a phosphorylation-deficient mutant, M2CXCR1, was constructed. This receptor, stably expressed in RBL-2H3 cells, coupled more efficiently to G protein and stimulated enhanced phosphoinositide hydrolysis, cAMP production, exocytosis, and phospholipase D activation, and was resistant to IL-8-induced receptor internalization. The rate and total amount of ligand stimulated actin polymerization remained unchanged, but interestingly, chemotaxis was decreased by approximately 30% compared with the wild type receptor. To study the role of receptor phosphorylation in cross-desensitization of chemoattractant receptors, M2CXCR1 was coexpressed with cDNAs encoding receptors for either fMLP (FR), C5a (C5aR), or platelet-activating factor (PAFR). Both C5aR and PAFR were cross-phosphorylated upon M2CXCR1 activation, resulting in attenuated guanosine 5'-3'-O-(thio)triphosphate (GTPgammaS) binding in membranes. In contrast, FR and M2CXCR1 were resistant to cross-phosphorylation and cross-inhibition of GTPgammaS binding by other receptors. Despite the resistance of M2CXCR1 to cross-phosphorylation and receptor/G protein uncoupling, its susceptibility to cross-desensitization of its Ca2+ response by fMLP and C5a, was equivalent to CXCR1. Regardless of the enhancement in certain receptor functions in M2CXCR1 compared with the wild type CXCR1, the mutated receptors mediated equivalent PLCbeta3 phosphorylation and cross-desensitization of Ca2+ mobilization by FR, C5aR, and PAFR. The results herein indicate that phosphorylation of CXCR1 regulates some, but not all of the receptors functions. While receptor phosphorylation inhibits G protein turnover, PLC activation, Ca2+ mobilization and secretion, it is required for normal chemotaxis and receptor internalization. Since phosphorylation of CXCR1 had no effect on its ability to induce phosphorylation of PLCbeta3 or to mediate class-desensitization, these activities may be mediated by independently regulated pathways.

Cross-desensitization Among Receptors for Platelet Activating Factor and Peptide Chemoattractants: EVIDENCE FOR INDEPENDENT REGULATORY PATHWAYS

Cross-desensitization among receptors for peptide chemoattractants have been shown to involve two independent processes, receptor phosphorylation and inhibition of phospholipase C (PLC) activation. Receptors for lipid chemoattractants, i.e. platelet activating factor (PAF) and leukotriene B4, did not inhibit the responses of peptide chemoattractant receptors, suggesting distinct signaling pathways. To examine cross-desensitization between receptors for lipid and peptide chemoattractants, cDNA encoding the PAF receptor (PAFR) was co-expressed into RBL-2H3 cells with cDNAs encoding receptors for either formylated peptides (FR), a product of the fifth component of complement (C5aR) or interleukin-8 A (IL-8RA). PAFR was homologously phosphorylated and desensitized by PAF, and cross-phosphorylated and cross-desensitized by fMet-Leu-Phe, C5a, and IL-8. In contrast, the receptors for peptide chemoattractants were neither cross-phosphorylated nor cross-desensitized by PAF. Staurosporine blocked cross-phosphorylation and cross-desensitization of the PAFR by peptide chemoattractants. Truncation of the cytoplasmic tail of PAFR (mPAFR) abolished its homologous and cross-phosphorylation. mPAFR was also resistant to cross-desensitization by peptide chemoattractants at the level of PLC activation. Interestingly, mPAFR mediated a sustained Ca2+ mobilization in response to PAF and was more active in inducing GTPase activity, phosphoinositide hydrolysis, secretion, and phospholipase D activation than the wild type PAFR. In contrast to PAFR, stimulation of the mPAFR cross-phosphorylated and cross-desensitized responses to IL-8RA. As expected, FR, which is resistant to cross-phosphorylation by C5aR and IL-8RA, was not phosphorylated by mPAFR. However, unlike C5aR and IL-8RA, mPAFR did not inhibit the ability of FR to activate PLC. Blocking Ca2+ influx inhibited mPAFR-mediated sustained Ca2+ response, phospholipase D activation and secretion, but not phosphoinositide hydrolysis and cross-phosphorylation and cross-desensitization of IL-8RA. The data herein suggest that cross-desensitization of PAFR by peptide chemoattractants is solely due to receptor phosphorylation. The PAFR and the peptide chemoattractant receptors do not cross-regulate each other at the level of PLC, suggesting distinct regulatory pathways.

Early inflammatory responses to Candida albicans infection in inbred and complement-deficient mice

Inflammatory responses that developed in the footpad during the first 48 h after inoculation of Candida albicans were compared in six genetically defined inbred strains of mice. Tissue responses consisted predominantly of accumulations of polymorphonuclear leucocytes, the magnitude of which was significantly less in mice lacking the fifth component of complement (C5). Despite this, there was no difference between C5-sufficient and C5-deficient mice in the total infectious burden, nor did depletion of complement by treatment with cobra venom factor cause any detectable reduction in the numbers of inflammatory cells in the area of the lesion. Ablation of granulocytes had no significant effect on the fungal burden over the period of the experiment. Immunisation provided some protection against tissue damage, but did not reduce the number of yeasts at the site of infection.

Strain-dependent differences in host response to Candida albicans infection in mice are related to organ susceptibility and infectious load

After systemic infection with the yeast Candida albicans, inbred mice show substantial differences in mortality, organ colonization, and severity of tissue damage. To examine the relationships between these variables, which are not directly correlated with each other, fungal colonization of the kidneys and brain was enumerated in six inbred strains that exhibit different patterns of tissue damage and mortality. Mice lacking the fifth component of complement (C5) are highly susceptible to lethal challenge, and A/J and DBA/2 mice, both C5 deficient, showed the highest colony counts in the kidneys after challenge with 10(5) blastoconidia. In contrast, colony counts in the brain of all six strains were equivalent at this challenge dose. A/J and DBA/2 mice died after challenge with 3 x 10(5) blastoconidia, but other strains showed an increase in kidney colonization, and strain-dependent differences in clearance from the brain became evident. The data suggest that mortality in A/J and DBA/2 mice is related to an unusual susceptibility of the kidneys to colonization by C. albicans and that there may be tissue-specific differences in host protective mechanisms.

B cells solicit their own help from T cells.

We have made use of T cell receptor (TCR)-transgenic mice with CD4+ T cells expressing a receptor specific for the self-antigen C5 (fifth component of complement) to study the role of different antigen-presenting cells in the determination of CD4+ T cell effector type. Contact of T cells from C5 TCR-transgenic mice with C5 protein or C5 peptide in vivo or in vitro induces biased T helper cell (Th) 1 type responses resulting in exclusive production of high levels of interferon gamma and interleukin (IL) 2. Transgenic mice, in contrast to nontransgenic littermates, do not generate an antibody response to C5. We show in this paper that B cell presentation in vitro induces a switch to the Th2 subset indicated by production of IL-4, and targetting C5 to B cells in vivo results in the generation of C5-specific antibodies.

Macrophage Presentation of Endogenous Self-Protein: The MHC Class II Presentation Pathway Is Not Accessible to Intracellular C5 or α1-Antitrypsin

This paper addresses the question of whether macrophages can present biosynthesized protein in a class II-restricted manner using the endogenous rather than the exogenous pathway of presentation. Two distinct self-antigens, the fifth component of complement (C5) and α1-antitrypsin, were studied. Both antigens are serum proteins synthesized by hepatocytes and macrophages. To direct synthesis exclusively to macrophages chimeras were constructed by transfer of bone marrow from donors expressing the self-antigen into irradiated hosts deficient for the respective self-antigen. Macrophages from such mice were unable to present biosynthesized C5 to class II-restricted T cells, even when preactivatedin vivo.While C5 production by macrophages is low and may not reach critical levels of intracellular protein required to access the class II presentation pathway, human α1-antitrypsin, expressed as a transgene in mice, was synthesized at 600-fold higher levels than C5. Nevertheless, macrophage-synthesized α1-antitrypsin in bone marrow chimeras was not presented in the context of class II—even in a mutant form which is sequestered in high amounts in the endoplasmic reticulum. We conclude that macrophages are unable to use the endogenous class II presentation pathway for these two model self-antigens. As a consequence MHC class II-restricted T cells specific for C5 and α1-antitrypsin remain ignorant of the presence of self-antigen within macrophages and are neither tolerized nor rendered autoimmune.

Mechanisms of tolerance induction in major histocompatibility complex class II-restricted T cells specific for a blood-borne self-antigen.

Transgenic mice expressing a major histocompatibility complex class II-restricted T cell receptor with specificity for a natural self-antigen, the fifth component of complement, were generated to analyze the mechanism of tolerance induction to a blood-borne self-protein. In the absence of C5 protein thymocytes from T cell receptor transgenic mice develop into mature CD4 single positive cells which emigrate into the periphery and mount C5-specific T cell responses upon immunization with C5. In the presence of circulating C5 protein, CD4 single positive thymocytes do not develop. Negative selection occurs late in thymic ontogeny leaving the bulk of CD4+8+ thymocytes unaffected. This phenotype may be due to a delay in contact with self-antigen presentation which, under physiological conditions, is inefficient in the cortex of C5+ mice, and therefore does not affect most immature double positive thymocytes. In contrast, in vitro exposure to C5(-)-presenting dendritic cells or in vivo injection of C5 peptide results in deletion of double positive thymocytes. C5+ transgenic mice are tolerant in vivo, but contain T cells in spleen and lymph nodes that secrete interleukin 2 and interferon gamma in response to C5 activation in vitro. When crossed onto a Rag1-/- background to prevent endogenous T cell receptor rearrangements, these peripheral potentially autoreactive cells do not appear. This indicates that endogenous T cell receptor rearrangements possibly leading to the expression of two receptors might be a prerequisite for their survival and export into the periphery.