Loxoprofen sodium induces the production of complement C5a in human serum.

Basophil activation test (BAT) is an in vitro allergy test that is useful to identify allergens that cause IgE-dependent allergies. The test has been used to detect not only food allergies and allergies caused by environmental factors but also to detect drug hypersensitivity, which has been known to include IgE-independent reactions. In our preliminary studies in which BAT was applied to detect hypersensitivity of loxoprofen, a non-steroidal anti-inflammatory drug (NSAID), conventional BAT with incubation for 30min did not show basophil activation by means of increased CD203c expression. In this study, we extended the incubation time to 24h on the basis of the hypothesis that loxoprofen indirectly activates basophils. Basophils from healthy control donors as well as allergic patients showed up-regulation of CD203c after incubation with loxoprofen for 24h. Activation was induced using loxoprofen-treated serum. Proteomic and pharmacologic analyses revealed that serum incubation with loxoprofen generated an active complement component C5a, which induced CD203c expression via binding to the C5a receptor on basophils. Because C3a production was also detected after incubation for 24h, loxoprofen is likely to stimulate the complement classical pathway. Our findings suggest that the complement activation is involved in drug hypersensitivity and the suppression of this activation may contribute to the elimination of false positive of BAT for drug allergies.

Improvement of prognostic performance in severely injured patients by integrated clinico-transcriptomics: a translational approach.

INTRODUCTION: Severe trauma triggers a systemic inflammatory response that contributes to secondary complications, such as nosocomial infections, sepsis or multi-organ failure. The present study was aimed to identify markers predicting complications and an adverse outcome of severely injured patients by an integrated clinico-transcriptomic approach. METHODS: In a prospective study, RNA samples from circulating leukocytes from severely injured patients (injury severity score ≥ 17 points; n = 104) admitted to a Level I Trauma Center were analyzed for dynamic changes in gene expression over a period of 21 days by quantitative RT-PCR. Transcriptomic candidates were selected based on whole genome screening of a representative discovery set (n = 10 patients) or known mechanisms of the immune response, including mediators of inflammation (IL-8, IL-10, TNF-α, MIF, C5, CD59, SPHK1), danger signaling (HMGB1, TLR2, CD14, IL-33, IL-1RL1), and components of the heme degradation pathway (HP, CD163, HMOX1, BLVRA, BLVRB). Clinical markers comprised standard physiological and laboratory parameters and scoring systems routinely determined in trauma patients. RESULTS: Leukocytes, thrombocytes and the expression of sphingosine kinase-1 (SPHK1), complement C5, and haptoglobin (HP) have been identified as markers with the best performance. Leukocytes showed a biphasic course with peaks on day 0 and day 11 after trauma, and patients with sepsis exhibited significantly higher leukocyte levels. Thrombocyte numbers showed a typical profile with initial thrombopenia and robust thrombocytosis in week 3 after trauma, ranging 2- to 3-fold above the upper normal value. 'Relative thrombocytopenia' was associated with multi-organ dysfunction, the development of sepsis, and mortality, the latter of which could be predicted within 3 days prior to the time point of death. SPHK1 expression at the day of admission indicated mortality with excellent performance. C5-expression on day 1 after trauma correlated with an increased risk for the development of nosocomial infections during the later course, while HP was found to be a marker for the development of sepsis. CONCLUSIONS: The combination of clinical and transcriptomic markers improves the prognostic performance and may represent a useful tool for individual risk stratification in trauma patients.

Human skin mast cells express complement factors C3 and C5.

We examine whether complement factor C3 or C5 is synthesized by human skin-derived mast cells and whether their synthesis is regulated by cytokines. C3 and C5 mRNAs were assessed by RT-PCR, and proteins by flow cytometry, confocal microscopy, Western blotting, and ELISA. C3 and C5 mRNAs were each expressed, and baseline protein levels/10(6) cultured mast cells were 0.9 and 0.8 ng, respectively, and located in the cytoplasm outside of secretory granules. C3 accumulated in mast cell culture medium over time and by 3 d reached a concentration of 9.4 ± 8.0 ng/ml, whereas C5 levels were not detectable (<0.15 ng/ml). Three-day incubations of mast cells with IL-1α, IL-1ß, IL-17, IFN-γ, IL-6, or anti-FcεRI did not affect C3 protein levels in culture medium, whereas incubations with PMA, TNF-α, IL-13, or IL-4 enhanced levels of C3 1.7- to 3.3-fold. In contrast with C3, levels of C5 remained undetectable. Importantly, treatment with TNF-α together with either IL-4 or IL-13 synergistically enhanced C3 (but not C5) production in culture medium by 9.8- or 7.1-fold, respectively. This synergy was blocked by attenuating the TNF-α pathway with neutralizing anti-TNF-α Ab, soluble TNFR, or an inhibitor of NF-κB, or by attenuating the IL-4/13 pathway with Jak family or Erk antagonists. Inhibitors of PI3K, Jnk, and p38 MAPK did not affect this synergy. Thus, human mast cells can produce and secrete C3, whereas ß-tryptase can act on C3 to generate C3a and C3b, raising the likelihood that mast cells engage complement to modulate immunity and inflammation in vivo.

Expression and characterization of the C345C/NTR domains of complement components C3 and C5.

Complement components C3, C4, and C5 are members of the thioester-containing alpha-macroglobulin protein superfamily. Within this superfamily, a unique feature of the complement proteins is a 150-residue-long C-terminal extension of their alpha-subunits that harbors three internal disulfide bonds. Previous reports have suggested that this is an independent structural module, homologous to modules found in other proteins, including netrins and tissue inhibitors of metalloproteinases. Because of its distribution, this putative module has been named both C345C and NTR. To assess the structures of these segments of the complement proteins, their relationships with other domains, and activities as independent structures, we expressed C345C from C3 and C5 in a bacterial strain that permits cytoplasmic disulfide bond formation. Affinity purification directly from cell lysates yielded recombinant C3- and C5-C345C with properties consistent with multiple intramolecular disulfide bonds and high beta-sheet contents. rC5-, but not rC3-C345C inhibited complement hemolytic activity, and surface plasmon resonance studies revealed that rC5-C345C binds to complement components C6 and C7 with dissociation constants of 10 and 3 nM, respectively. Our results provide strong evidence that this binding corresponds to the previously described reversible binding of C5 to C6 and C7, and taken together with earlier work, indicate that the C5-C345C module interacts directly with the factor I modules in C6 and C7. The high binding affinities suggest that complexes composed of C5 bound to C6 or C7 exist in plasma before activation and may facilitate assembly of the complement membrane attack complex.

Formation of high affinity C5 convertase of the classical pathway of complement.

C3/C5 convertase is a serine protease that cleaves C3 and C5. In the present study we examined the C5 cleaving properties of classical pathway C3/C5 convertase either bound to the surface of sheep erythrocytes or in its free soluble form. Kinetic parameters revealed that the soluble form of the enzyme (C4b,C2a) cleaved C5 at a catalytic rate similar to that of the surface-bound form (EAC1,C4b,C2a). However, both forms of the enzyme exhibited a poor affinity for the substrate, C5, as indicated by a high Km (6-9 microM). Increasing the density of C4b on the cell surface from 8,000 to 172,000 C4b/cell did not influence the Km. Very high affinity C5 convertases were generated only when the low affinity C3/C5 convertases (EAC1,C4b,C2a) were allowed to deposit C3b by cleaving native C3. These C3b-containing C3/C5 convertases exhibited Km (0.0051 microM) well below the normal concentration of C5 in blood (0.37 microM). The data suggest that C3/C5 convertase assembled with either monomeric C4b or C4b-C4b complexes are inefficient in capturing C5 but cleave C3 opsonizing the cell surface with C3b for phagocytosis. Deposition of C3b converts the enzymes to high affinity C5 convertases, which cleave C5 in blood at catalytic rates approaching Vmax, thereby switching from C3 to C5 cleavage. Comparison of the kinetic parameters with those of the alternative pathway convertase indicates that the 6-9-fold greater catalytic rate of the classical pathway C5 convertase may compensate for the fewer numbers of C5 convertase sites generated upon activation of this pathway.

Expression of complement 3 and complement 5 in newt limb and lens regeneration.

Some urodele amphibians possess the capacity to regenerate their body parts, including the limbs and the lens of the eye. The molecular pathway(s) involved in urodele regeneration are largely unknown. We have previously suggested that complement may participate in limb regeneration in axolotls. To further define its role in the regenerative process, we have examined the pattern of distribution and spatiotemporal expression of two key components, C3 and C5, during limb and lens regeneration in the newt Notophthalmus viridescens. First, we have cloned newt cDNAs encoding C3 and C5 and have generated Abs specifically recognizing these molecules. Using these newt-specific probes, we have found by in situ hybridization and immunohistochemical analysis that these molecules are expressed during both limb and lens regeneration, but not in the normal limb and lens. The C3 and C5 proteins were expressed in a complementary fashion during limb regeneration, with C3 being expressed mainly in the blastema and C5 exclusively in the wound epithelium. Similarly, during the process of lens regeneration, C3 was detected in the iris and cornea, while C5 was present in the regenerating lens vesicle as well as the cornea. The distinct expression profile of complement proteins in regenerative tissues of the urodele lens and limb supports a nonimmunologic function of complement in tissue regeneration and constitutes the first systematic effort to dissect its involvement in regenerative processes of lower vertebrate species.

In vitro complement activation favoring soluble C5b-9 complex formation alters myocellular sodium homeostasis.

BACKGROUND: Deranged Na(+) homeostasis in skeletal muscle is closely associated with excessive complement activation that is encountered during sepsis. Recent evidence suggests that soluble C5b-9 complexes (SC5b-9), which are readily detected in plasma during sepsis and have long been considered irrelevant nonmembrane binding end products of complement activation, may have numerous biologic effects. The purpose of this study, therefore, was to determine the effects of SC5b-9 on myocellular ion homeostasis and its mechanism(s) of action. METHODS: Hindlimb fast-twitch extensor digitorum longus (EDL) was freshly isolated from rats weighing 50 to 70 g and then incubated at 30 degrees C for 60 minutes in normal Krebs-Henseleit buffer (KHB, pH 7.4) containing 10% zymosan-activated rat serum (10 mg/mL at 37 degrees C for 60 minutes) as a source of SC5b-9. Zymosan particles were removed by centrifugation after activation to exclude any noncomplement direct effects. Heat-inactivated rat serum (56 degrees C for 30 minutes) was used as control. EDL muscle was also incubated with pertussis toxin (1 microg/mL), in Ca(2+)-free KHB, with thapsigargin (0.3 or 3 micromol/L), or with ouabain (0.01, 0.1 or 1 micromol/L) before and/or during incubation with 10% zymosan-activated or heat-inactivated rat serum. Intracellular Na(+) and K(+) contents ([Na(+)](i) or [K(+)](i)) of EDL muscle were determined by using flame photometry after washing in ice-cold Na(+)-free Tris-sucrose buffer. SC5b-9 in zymosan-activated human serum was determined by SC5b-9 enzyme-linked immunoassay. RESULTS: SC5b-9 in zymosan-activated human serum significantly increased by 400% as compared with nonactivated, normal human serum. Zymosan-activated rat serum markedly increased [Na+]i without affecting [K(+)](i) in fast-twitch EDL muscle, which was completely inhibited by pertussis toxin, removal of extracellular Ca(2+) or depletion of intracellular Ca(2+) with thapsigargin. The addition of ouabain (at micromolar concentrations) increased myocellular [Na(+)](i) and decreased myocellular [K(+)](i) in both the zymosan-activated and the heat-inactivated rat serum groups. The effects of ouabain on myocellular [Na(+)](i) and [K(+)](i) were equivalent in these 2 groups. Zymosan-activated and heat-inactivated rat serum had similar effects on myocellular [K(+)](i) in the presence or absence of pertussis toxin, removal of extracellular Ca(2+) or depletion of intracellular Ca(2+). CONCLUSIONS: Zymosan-activated rat serum (presumed SC5b-9 enriched) selectively alters Na(+) homeostasis in isolated fast-twitch skeletal muscle. The mechanisms for such effects may be linked to G-proteins, Ca(2+) flux and Na(+),K(+)-adenosine triphosphatase pump binding site blockade.

Generation of complement C3 and expression of cell membrane complement inhibitory proteins by human bronchial epithelium cell line.

BACKGROUND: The interrelationship between human airway epithelium and complement proteins may affect airway defence, airway function, and airway epithelial integrity. A study was undertaken to determine (1) whether unstimulated human bronchial epithelium generates complement proteins and expresses cell membrane complement inhibitory proteins (CIP) and (2) whether stimulation by proinflammatory cytokines affects the generation of complement and expression of cell membrane CIP by these cells. METHODS: Human bronchial epithelium cell line BEAS-2B was cultured in a serum-free medium. Cells were incubated with and without proinflammatory cytokines to assess unstimulated and stimulated generation of complement C3, C1q and C5 (by ELISA), and to examine the expression of cell membrane CIP decay accelerating factor (DAF; CD55), membrane cofactor protein (MCP; CD46), and CD59 (protectin) by flow cytometry analysis. RESULTS: Unstimulated human bronchial epithelial cell line BEAS-2B in serum-free medium generates complement C3 (mean 32 ng/10(6) cells/72 h, range 18-52) but not C1q and C5, and expresses cell membrane DAF, MCP, and CD59. Interleukin (IL)-1alpha (100 U/ml/72 h) and tumour necrosis factor (TNF-alpha; 1000 U/ml/72 h) increased generation of C3 up to a mean of 78% and 138%, respectively, above C3 generation by unstimulated cells. DAF was the only cell membrane CIP affected by cytokine stimulation. Interferon (IFN)-gamma (10 U/ml/72 h) and TNF-alpha (1000 U/ml/72 h) increased DAF expression up to a mean of 116% and 45%, respectively, above that in unstimulated cells. MCP and CD59 expression was not consistently affected by IL-1alpha, TNF-alpha, or IFN-gamma. CONCLUSIONS: Local generation of complement C3 and expression of cell membrane CIP by human bronchial epithelium and its modulation by proinflammatory cytokines might be an additional regulatory mechanism of local airway defence and may affect airway function and epithelial integrity in health and disease.

rC5a directs the in vitro migration of human memory and naive tonsillar B lymphocytes: implications for B cell trafficking in secondary lymphoid tissues.

Human C5a is a potent chemoattractant for granulocytes, monocytes, and dendritic cells. In mice C5a has been shown to be chemotactic for germinal center (GC) B cells. To date, no information is available on the effects of C5a on human B cell locomotion. Here we demonstrate that rC5a increases polarization and migration of human tonsillar B cells. The locomotory response was due to both chemokinetic and chemotactic activities of rC5a. Moreover, memory and, at a lesser extent, naive B cell fractions from purified tonsillar populations displayed rC5a-enhanced migratory properties, whereas GC cells did not. Flow cytometry revealed C5aR (CD88) on approximately 40% memory and 10% naive cells, respectively, whereas GC cells were negative. Immunohistochemistry showed that a few CD88+ cells were of the B cell lineage and localized in tonsillar subepithelial areas, where the majority of memory B cells settle. Pretreatment of memory B cells with the CD88 mAb abolished their migratory responsiveness to rC5a. Finally, the C5 gene was found to be expressed in naive, GC, and memory B lymphocytes at both the mRNA and the protein level. This study delineates a novel role for C5a as a regulator of the trafficking of human memory and naive B lymphocytes and supports the hypothesis that the B cells themselves may serve as source of C5 in secondary lymphoid tissues.

Excessive degradation of intracellular protein in macrophages prevents presentation in the context of major histocompatibility complex class II molecules.

The endogenous major histocompatibility complex (MHC) class II presentation pathway allows biosynthesized, intracellular antigens access for presentation to MHC class II-restricted T cells. This pathway has been well documented in B cells and fibroblasts, but may not be universally available in all antigen-presenting cell types. This study compares the ability of different antigen-presenting cells, expressing endogenous C5 protein (fifth component of mouse complement) as a result of transfection, to present their biosynthesized C5 to MHC class II-restricted T cells. B cells and fibroblasts expressing C5 were able to present several epitopes of this protein with MHC class II molecules, whereas macrophages were unable to do so, but readily presented C5 from an extracellular source. However, macrophage presentation of endogenous C5 could be achieved when they were treated with low doses of the lysosomotropic agent ammonium chloride. In the presence of an inhibitor of autophagy, presentation of endogenous C5 was abrogated, indicating that biosynthesized C5 is shuttled into lysosomal compartments for degradation before making contact with MHC class II molecules. Taken together, this suggests that proteolytic activity in lysosomes of macrophages may be excessive, compared with fibroblasts and B cells, and destroys epitopes of the C5 protein before they can gain access to MHC class II molecules. Thus, there are inherent differences in presentation pathways between antigen-presenting cell types; this could reflect their specialized functions within the immune system with macrophages focussing preferentially on internalization, degradation, and presentation of extracellular material.

Complement activation by myeloperoxidase products released from stimulated human polymorphonuclear leukocytes.

Purified human myeloperoxidase (MPO) converted human C5 to an activated form, i.e. the C5 protein adopted a configuration expressing a binding site for C6; the resulting C56 complex then reacted with C7, C8 and C9 forming a hemolytic C5-9 complex. For the activation by myeloperoxidase chloride and hydrogen peroxide were essential. This indicates that the peroxidase acted through the generation of HOCl which had been shown earlier to oxidize and activate C5. Human polymorphonuclear leukocytes (PMN) were stimulated in vitro by incubation with opsonized zymosan; thereafter the supernatants were tested for C5 activating potency. Stimulated PMN release H2O2 and MPO that produces hypochlorite and secondarily various chloramines. As a trap for the labile hypochlorite generated excess taurine was added to the PMN suspensions during the incubation. Hypochlorite is then stoichiometrically converted to the relatively stable taurine chloramine. In order to rule out interfering activities of proteolytic enzymes released from the PMN and known to attack C5, the supernatants were ultracentrifuged, and the ultrafiltrates, containing only low molecular weight compounds, were used for the further studies. They contained taurine chloramine, estimated photometrically, and they activated C5 upon incubation, assayed functionally by reactive lysis. Azide, an inhibitor of myeloperoxidase, and catalase which destroys H2O2, essential for MPO-catalyzed oxidations, prevented the generation of C5 activating potency and of chloramines. Unstimulated PMN produced neither oxidants nor C5 activating potency. When taurine was omitted from the PMN suspensions during stimulations much less oxidant was found in the supernatants and less C5 activating potency. These findings indicate that the C5 activating agent was produced by stimulated PMN through MPO-generated hypochlorite, trapped as taurine chloramine. In the absence of added taurine the hypochlorite formed by MPO oxidized endogenous amines that also activated C5. Further studies suggested that among these was some monochloramine derived from endogenous ammonia. Activation of the terminal complement reaction sequence by MPO released from stimulated PMN may represent a third pathway to complement activation contributing to and reinforcing complement and PMN functions at the site of inflammation or tissue injury.

Macrophage presentation of endogenous self-protein: the MHC class II presentation pathway is not accessible to intracellular C5 or alpha 1-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 alpha 1-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 preactivated in 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 alpha 1-antitrypsin, expressed as a transgene in mice, was synthesized at 600-fold higher levels than C5. Nevertheless, macrophage-synthesized alpha 1-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 alpha 1-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.

Silica induced suppression of the production of third and fifth components of the complement system by human lung cells in vitro.

Although investigations to date have demonstrated the ability of the monocyte/macrophage to synthesize complement components, only a limited number of studies on complement synthesis by nonhepatic tissue cells have been reported. To begin to fill this gap in our knowledge we have recently evaluated the ability of lung tissue cells to synthesize and secrete various complement components in vitro. Using 35S-methionine incorporation and immunoprecipitation techniques we have previously demonstrated the ability human lung type II pneumocytes (A549) and human lung fibroblasts (WI-38), to synthesize and secrete a variety of both early and terminal complement components, as well as several regulatory proteins including Clr, Cls, C4, C3, C5, C6, C7, C8, C9, Factor B, Factor H, Factor I and Cls inactivator. Our present studies demonstrate the capability of silica to regulate complement component production by A549 cells, but not complement component production by WI-38 cells. Specifically, using sensitive ELISAs we demonstrated that a non-toxic dose of silica had the capability to suppress the production of both C3 and C5 by A549 pneumocytes by 40-50 percent, but had no effect on C3 or C5 synthesis by WI-38 fibroblasts. Additionally, using 35S-methionine incorporation and TCA precipitation techniques, we demonstrated that suppression of C3 and C5 production by silica treated A549 pneumocytes was not a result of suppression of total protein synthesis. These studies demonstrate that silica, which has been implicated in pulmonary diseases, has the capability to regulate local complement production by lung tissue cells in vitro. In vivo, this suppression of complement production by the type II pneumocytes could alter the local tissue reservoir of complement components during infection and pulmonary injury, thus resulting in depressed pulmonary host defense.

Study on the synthesis of complement components by human renal mesangial cells in culture. Assessment of the effect of cytokines.

Renal mesangial cell (MC) cultures are easily established and widely used. MC produce some complement (C) regulatory proteins. We studied whether MC synthesize C components (C3, C5, C8). MC cultures were established from normal portions of cortices of nephrectomies for renal cancer. After growing to near-confluence in RPMI/17% FBS and resting for 24 h in RPMI/0.5% FBS, MC were stimulated up to 72 h with IL-1 beta or IL-6 (10, 100, 1000 U/ml). Neither C5 nor C8 were detected by ELISA. While C3 was present in supernatant under basal conditions (15.5-107.6 ng/10(6) cells/24h) in different MC lines. IL-1 beta up-regulated the synthesis by 2.4-4.5 folds, whereas IL-6 did not show any effect. C3 synthetic rate was 1.76 ng/h/10(6) cells under IL-1 stimulation versus basal rate of 0.37 ng/h/10(6) cells. MC production of C3, especially induced by IL-1 may have pathogenetic relevance in glomerulonephritis.

Localization of self antigen: implications for antigen presentation and induction of tolerance.

The fifth component of complement (C5) is a self antigen expressed in serum of normal mice at a concentration of about 50 micrograms/ml. We have previously shown that C5 is constitutively processed and presented by antigen-presenting cells (APC) in normal mice to induce and maintain complete tolerance in major histocompatibility complex (MHC) class II-restricted T cells. This report addresses the question of whether C5 presentation involves exogenous antigen which has been internalized for processing or whether intracellular, biosynthesized C5 is being presented with MHC class II. Macrophages were found to synthesize, but not secrete C5 in bone marrow chimeras made from irradiated C5-deficient [C5(-)] hosts reconstituted with C5-sufficient [C5(+)] bone marrow [C5(+)-->C5(-)]. In these mice, macrophages are the only source of C5. [C5(+)-->C5(-)] chimeras are not tolerant of C5 and generate C5-specific T and B cell responses upon immunization indistinguishable from those of C5(-) mice. Macrophages from [C5(+)-->C5(-)] chimeras are unable to activate C5-specific T cell hybrids in vitro unlike macrophages from a C5(-) strain that has matured in a C5-expressing environment [C5(-)-->C5(+) chimeras]. This shows that under physiological conditions in vivo intracellular C5 does not get access to the class II presentation pathway and thus, does not induce tolerance in class II-restricted T cells.

Cytokine regulation of C3 and C5 production by human corneal fibroblasts.

Recent investigations have suggested that cytokines play important roles during inflammation and host defense, primarily by regulating the diverse functions of immunologic cells (e.g. lymphocytes and monocytes). However, much less is known about the capacity of cytokines to also regulate the functions of resident tissue cells. We hypothesize that during inflammation, cytokines (e.g. monokines and lymphokines) directly regulate the expression of inflammatory precursors and mediators, such as the third and fifth complement components, by resident ocular cells and are therefore important in the local regulation of ocular inflammation. To test this hypothesis we developed an in vitro culture system utilizing isolated human corneal fibroblasts and examined the effects of specific cytokines, i.e. interleukins and interferons, on the production of the third and fifth components of the complement system. Human corneal fibroblasts were cultured in the presence of varying concentrations (1-500 U ml-1) of interleukin 1 alpha, interleukin 1 beta, interleukin 2, interferon alpha and interferon gamma for 48 hr at 37 degrees C, 5% CO2. The supernatants were then evaluated for antigen levels for the third and fifth components of complement using specific enzyme-linked immunospecific assays. These studies revealed that both interleukin 1 alpha and interleukin 1 beta induced seven to tenfold increases in the levels of the third component. Similarly interferon alpha and interferon gamma stimulated an approximate four and ninefold dose-dependent increase, respectively, in the production of the third component. Analysis of the effect of interleukin 2 on third component production demonstrated that higher concentrations (100 U ml-1) were required to induce a fivefold increase in the production of the third component.(ABSTRACT TRUNCATED AT 250 WORDS)

Human umbilical vein endothelial cells synthesize functional C3, C5, C6, C8 and C9 in vitro.

Human endothelial cells (EC), cultured serum-free, synthesize de novo protein which increasingly bind to agarose beads (an alternative pathway activator), until a plateau phase is reached after 24-48 h. EC synthesize functional C3, C5, C6, C8 and C9, which were detected on co-cultured agarose beads, using relevant polyclonal anti-complement antibodies. Two monoclonal anti-C9 neoepitope antibodies (aE11, poly C9-MA) bound to the co-cultured beads, showing that the terminal complement complex (TCC) (C5b-9) was assembled on the beads. This also suggests that C7 is synthesized. There seems to be a positive correlation between the amount of agarose-bound labelled protein and agarose-bound complement. The results indicate that EC produce and secrete the components for the functional alternative and terminal pathways of complement.

Chromosomal locations and gonadal dependence of genes that mediate resistance to ectromelia (mousepox) virus-induced mortality.

Four genetic loci were tested for linkage with loci that control genetic resistance to lethal ectromelia virus infection in mice. Three of the loci were selected because of concordance with genotypes assigned to recombinant inbred (RI) strains of mice derived from resistant C57BL/6 and susceptible DBA/2 (BXD) mice on the basis of their responses to challenge infection. Thirty-six of 167 male (C57BL/6 x DBA/2)F1 x DBA/2 backcross (BC) mice died (22%), of which 27 (75%) were homozygous for DBA/2 alleles at Hc and H-2D. Twenty-eight percent of sham-castrated and 6% of sham-ovariectomized BC mice were susceptible to lethal mousepox, whereas 50% of gonadectomized mice were susceptible. There was no linkage evident between Hc or H-2D and loci that controlled resistance to lethal ectromelia virus infection in 44 castrated BC mice. Mortality among female mice of BXD RI strains with susceptible or intermediate male phenotypes was strongly correlated (r = 0.834) with male mortality. Gonadectomized C57BL/6 mice were as resistant as intact mice to lethal ectromelia virus infection. These results indicate that two gonad-dependent genes on chromosomes 2 and 17 and one gonad-independent gene control resistance to mousepox virus infection, that males and females share gonad-dependent genes, and that the gonad-independent gene is fully protective.

Binding of iron to the 5th component of human complement directs oxygen radical-mediated conversion to specific sites and causes nonenzymic activation.

It was earlier found that hydroxyl radicals (OH.) generated in a fluid phase system, convert human C5 to an activated, C5b-like form without cleavage. For the conversion catalytic amounts of divalent iron were necessary; upon their oxidation OH. are generated. In view of the extraordinary reactivity of OH. their specific effect on C5 was surprising. It has now turned out that C5 binds iron, about 13-15 Fe ions per molecule C5. C5 was fully bound to iron-loaded Chelating Sepharose and in this way removed form the medium. Only at the sites where iron is bound, is OH. generation possible. The half-life of OH. is extremely short and hence the radicals can act on C5 only in the immediate environment of the binding sites for iron. In this way the oxidative change of C5, induced by the radicals, is restricted to some sites on the molecule determined by the iron binding. The hydroxyl radical scavenger, dimethylsulfoxide (DMSO), is unable to interfere with the conversion of C5 by OH. generated on iron-Sepharose, it does not reach the site of reaction in that system. In fluid phase systems DMSO does inhibit the conversion of C5 to C5b-like C5.