Chemotactic factor inactivator interaction with Gc-globulin (vitamin D-binding protein). A mechanism of modulating the chemotactic activity of C5a

Chemotactic factor inactivator (CFI) can decrease the neutrophil chemotactic activity of C5a. Gc-Globulin (GcG) can function as a cochemotaxin for C5a by binding to C5a or C5a des Arg and enhancing its chemotactic potency. We hypothesized that CFI might interact with GcG and thus decrease the chemotactic activity of C5a. CFI was found to markedly inhibit the neutrophil chemotactic activity of partially purified C5a containing GcG (p less than 0.01). Addition of GcG was able to reverse the capacity of CFI to inhibit C5a-directed neutrophil chemotaxis (p less than 0.01). CFI had no significant effect on neutrophil chemotaxis when incubated with C5a depleted of GcG or C5a des Arg. CFI was also able to inhibit the interaction of C5a with GcG adsorbed to plastic. To determine if CFI interacted with GcG, a sandwich ELISA was used. These ELISA tests demonstrated that CFI directly interacted with GcG in a dose-dependent manner that was both heat and pH sensitive. To investigate the possibility of enzymatic degradation of C5a by CFI, CFI preparations were analyzed for carboxypeptidase activity, aminopeptidase activity, and for the capacity to cleave dansylated C5a. No enzymatic activity or cleavage was observed. Furthermore, the direct interaction of CFI with C5a and C5a des Arg was assessed by ELISA tests and column chromatography and no interaction was observed. These results suggest that CFI modulates C5a-directed neutrophil chemotaxis by interacting with GcG and preventing GcG from enhancing the chemotactic potency of C5a.     

Human C5a and C5a des Arg exhibit chemotactic activity for fibroblasts

C5a and C5a des Arg are chemotactic factors for inflammatory cells but it is not known whether these agents are chemoattractants for fibroblasts. Accordingly, C5a, purified from zymosan-activated human, and C5a des Arg, prepared by incubating C5a with immobilized porcine carboxypeptidase B, were studied for fibroblast chemotactic activity. We observed that both C5a and C5a des Arg stimulated human skin fibroblasts and fetal bovine ligament fibroblasts to migrate in a concentration-dependent fashion, and that the migratory responses were similar in magnitude to the responses achieved with optimal concentrations of two known fibroblast chemoattractants, platelet-derived growth factor and human fibrinopeptide B. The peak responses to C5a and C5a des Arg occurred at approximately 10(-9)M. With ligament fibroblasts, there was a greater response to C5a des Arg than to C5a, but with human fibroblasts there was no difference. Cochemotaxin, which enhances the chemotactic activity of C5a des Arg for neutrophils, had no effect on C5a des Arg fibroblast chemotactic activity but appeared to increase the fibroblast chemotactic activity of C5a. These results indicate that the effects of C5a and C5a des Arg in vivo may extend to the recruitment of mesenchymal cells. Moreover, the findings represent another example of an activity retained by C5a after removal of its carboxyl terminal arginine.     

Platelet-derived thrombospondin-1 is necessary for the vitamin D-binding protein (Gc-globulin) to function as a chemotactic cofactor for C5a

The chemotactic activity of C5a and C5a des Arg can be enhanced significantly by the vitamin D-binding protein (DBP), also known as Gc-globulin. DBP is a multifunctional 56-kDa plasma protein that binds and transports several diverse ligands. The objective of this study was to investigate the mechanisms by which DBP functions as a chemotactic cofactor for C5a using neutrophils and U937 cells transfected with the C5aR (U937-C5aR cells). The results demonstrate that U937-C5aR cells show C5a chemotactic enhancement only to DBP in serum, but, unlike mature neutrophils, this cell line cannot respond to DBP in plasma or to purified DBP. Analysis by SDS-PAGE and isoelectric focusing revealed no structural difference between DBP in serum compared with DBP in plasma. However, plasma supplemented with either serum, DBP-depleted serum, or activated platelet releasate provides a required factor and permits DBP to function as a chemotactic cofactor for C5a. Fractionation of activated platelet releasate revealed that the additional factor possessed the properties of thrombospondin-1 (TSP-1). Finally, purified TSP-1 alone could reproduce the effect of serum or platelet releasate, whereas Abs to TSP-1 could block these effects. These results provide clear evidence that TSP-1 is needed for DBP to function as a chemotactic cofactor for C5a.     

Factor XIII-dependent generation of 5th complement component(C5)-derived monocyte chemotactic factor coinciding with plasma clotting.

Blood coagulation or plasma clotting caused generation of a monocyte chemotactic factor(s) in vitro. The chemotactic factor, of which the apparent molecular mass was 75 kDa, shared antigenicity with complement C5 and possessed the affinity to monocytes, but not to polymorphonuclear leukocytes. The generation of the chemotactic factor was hindered in the presence of a thiol enzyme inhibitor, p-chloromercuriphenyl sulfonic acid, at the concentration of 1 mmol/l, although the gelation of plasma was apparently completed. Furthermore, the generation of chemotactic factor was not observed when a plasma deficient in blood coagulation factor XIII, which is a precursor of a thiol enzyme, plasma transglutaminase, was used; and the activity normally appeared when the deficient plasma was reconstituted with purified factor XIII or with a tissue transglutaminase prior to clotting. When the human sera were injected into guinea pig skin, the serum derived from normal plasma or from the reconstituted factor XIII deficient one caused mononuclear cell infiltration, however, the serum from the deficient plasma without reconstitution infiltrated to a significantly smaller extent. These results indicated that the complement system was initiated somehow during the clotting process resulting in the generation of the C5-derived monocyte chemotactic factor in cooperation with factor XIIIa (activated factor XIII).     

Residual hemolytic and proteolytic activity expressed by Bb after decay-dissociation of C3b,Bb

Bb (Mr = 63,000) is the catalytic site-bearing subunit of the C3 convertase of the alternative complement pathway, C3b,Bb, which is dissociated from the complex upon decay of the enzyme. Because purified Bb induced certain leukocyte activities, we examined whether it expresses residual hemolytic or proteolytic activity. Hemolytic activity of Bb was tested by using Factor B- or Factor D-depleted normal human serum and rabbit or sheep erythrocytes. Proteolytic activity of Bb was assessed by using purified C3 or C5 as substrates and SDS-PAGE to detect protein cleavage. Bb expressed metal-dependent hemolytic activity that was approximately 100-fold lower than that of Factor B. This activity could be inhibited by Factor H and enhanced by properdin. Low but statistically significant binding of 125I-labeled Bb to C3b on erythrocytes was demonstrated. Monoclonal antibodies that bind to Bb but not to intact Factor B inhibited the Bb hemolytic activity. Purified Bb cleaved C3 to C3a and C3b, as evidenced by the appearance of the alpha'-chain of C3b. It also cleaved C5 to C5a and C5b when cobra venom factor was present in the reaction mixture. Metal ions were required for expression of proteolytic activity, and Ni supported the activity better than Mg. These results indicate that decayed Bb has residual C3 and C5 cleaving activity and hemolytic activity, expression of which appears to require its association with C3b, C3(H2O), or cobra venom factor. These observations may aid in explaining the mechanism of action of Bb on leukocytes.     

Chemotactic peptides modulate adherence of human polymorphonuclear leukocytes to monolayers of cultured endothelial cells

We have used a new centrifugation assay to examine the effects of highly purified human C5a and C5a des Arg, as well as effects of N-formyl-methionyl-leucyl-phenylalanine (FMLP), on both the extent and strength of human polymorphonuclear leukocyte (PMN) adherence to monolayers of cultured human umbilical vein endothelial cells. At concentrations that were chemotactic for PMN, C5a (0.1 nM), C5a des Arg (5.0 nM), and FMLP (1.0 nM) significantly reduced the percentage of PMN that adhered to endothelial monolayers. Adherence also was reduced by C5a des Arg that was generated by incubating (37 degrees C, 30 min) fresh human serum with either zymosan or purified C5a. High concentrations of C5a (greater than 1.0 nM) and FMLP (greater than 50 nM) that diminished PMN chemotaxis significantly enhanced the percentage of PMN that adhered tightly to endothelial cells (adherent cells resisted a dislodgment force of 1200 X G). Tight adherence of PMN to endothelial cells also was increased by high concentrations of C5a that were added to human serum in which carboxypeptidase N activity was destroyed by heating (56 degrees C, 30 min), and by C5a that was generated by incubating (37 degrees C, 30 min) fresh human serum with zymosan in the presence of the carboxypeptidase N inhibitor, epsilon-aminocaproic acid. High concentrations of C5a des Arg (up to 80 nM) neither enhanced adherence of PMN to endothelial cells nor decreased PMN migration. Thus, a reciprocal relation exists between PMN migration and PMN adherence to endothelial cells in response to chemotactic factors. At concentrations that are chemotactic for human PMN, C5-derived peptides and FMLP reduce the adherence of PMN to endothelial monolayers. Only at concentrations that decrease PMN migration do C5a and FMLP augment PMN adherence.     

Substituted isocoumarins as inhibitors of complement serine proteases.

Inhibition of complement proteins D, B, C2, C1s, C1r, I, and the catalytic fragments Bb and C2a by substituted isocoumarins was investigated. 3,4-Dichloroisocoumarin, a general serine protease inhibitor, inhibited factor D, C1r, and C1s moderately with second-order inhibition constants (kobs/[I]) of 40 to 190 M-1 s-1, but it did not inhibit C2, factor B, C2a, or Bb. The best inhibitor for factors D and B was 4-chloro-7-guanidino-3-methoxyisocoumarin with kobs/[I] values of 250 and 290 M-1 s-1, respectively. Most isocoumarins did not inhibit C2 or C2a; only 4-chloro-3-isothiureidoalkoxyisocoumarins were slightly inhibitory. 3-Alkoxy-4-chloro-7-guanidinoisocoumarins inhibited C1r and C1s moderately. The best inhibitor for C1r and C1s was 4-chloro-3-(3-isothiureidopropoxy)isocoumarin with kobs/[I] values of 6,600 and 130,000 M-1 s-1, respectively. Fifty amino acid or peptide thioesters containing Arg or other amino acids at the P1 site were tested as substrates of factor I, however none was hydrolyzed. Isocoumarins substituted with chloro and basic groups such as guanidino and isothiureidoalkoxy inhibited factor I activity with its natural substrate C3b, but kobs/[I] values were low. 4-Chloro-3-ethoxy-7-guanidinoisocoumarin inhibited activation of the alternative pathway and, to a lesser extent, of the classical pathway in serum. Several other substituted isocoumarins also inhibited cobra venom factor-initiated activation of the alternative pathway in serum.     

Purification, cloning, and expression of a novel salivary anticomplement protein from the tick, Ixodes scapularis

The alternative pathway of complement is an important defense against pathogens and in tick rejection reactions. The tick Ixodes scapularis is able to feed repeatedly on its natural host and has a salivary anticomplement activity that presumably facilitates feeding. In this study, we purified and then obtained the amino-terminal sequence of the I. scapularis salivary anticomplement (Isac). We found a full-length clone coding for Isac by random screening of a salivary gland cDNA library. Expressing Isac cDNA in COS cells reproduced the activity found in tick saliva, namely, inhibition of rabbit erythrocyte lysis by human serum in the presence of Mg(2+) and EGTA, inhibition of C3b binding to agarose in the presence of Mg(2+) and EGTA, and acceleration of factor Bb uncoupling from the C3 convertase generated by the alternative pathway. Recombinant Isac had no effect on the recalcification time of human platelet-poor plasma or in the classical complement pathway, indicating that it is a specific inhibitor similar to the regulators of complement activation of the alternative pathway such as factor H. Isac, however, has no similarity to any protein in the GenBank(TM) data base, indicating that it is a novel and relatively small (18.5 kDa) anticomplement molecule.     

Absence of induction of IL-1 production in human monocytes by complement fragments

The ability of C fragments to induce IL-1 production in human monocytes was examined by using various approaches to carefully exclude the role of contaminating endotoxin. The presence of IL-1 activity in monocyte supernatants and lysates was assayed by the augmentation of PHA-induced proliferation of murine thymocytes. SRBC were opsonized with IgM rabbit antibodies and various human C components to prepare EAC reagents that contained less than 25 pg LPS/ml of EAC at 5 x 10(8) cells/ml. EAC1q, EAC4b, EAC4b2aoxy, EAC4b2aoxy C3b, EAC4b2aoxyC3bi, and EAC4b2aoxyC3d all failed to induce IL-1 production when incubated at 10- to 100-fold excess with adherent human monocytes. Similarly, LPS-free purified C3a, C5a, and C5a des Arg all showed no IL-1-inducing activities at concentrations up to 25 micrograms/ml. However, the same C5a preparations were active on human monocytes in the induction of chemotaxis, and C3a and C5a both induced skin-blueing in guinea pigs. Fragment Ba and Bb preparations purified by gel filtration chromatography contained approximately 100 pg LPS/micrograms Ba or Bb. These Ba and Bb preparations at 10 and 50 micrograms/ml, respectively, induced IL-1 production in the presence of 5 micrograms/ml polymyxin B (PMB). However, Ba and Bb preparations purified by affinity chromatography and HPLC contained lower levels of endotoxin contamination and displayed IL-1-inducing activities at Ba and Bb concentrations of 50 and 100 micrograms/ml, respectively, that were almost completely inhibited by PMB. To explore further the role of contaminating endotoxin, a Bb preparation was adsorbed with PMB-4B in the presence of a dialyzable detergent to remove LPS bound to the Bb. This LPS-free Bb preparation failed to induce IL-1 production while maintaining intact enzymatic activities. These results indicate that various solid phase or soluble C fragments, including C3b, iC3b, C3d, C3a, C5a, Ba or Bb do not induce IL-1 production in human monocytes in the absence of contaminating endotoxin.     

Structure and function of recombinant cobra venom factor

Cobra venom factor (CVF) is the complement-activating protein from cobra venom. It is a structural and functional analog of complement component C3. CVF functionally resembles C3b, the activated form of C3. Like C3b, CVF binds factor B, which is subsequently cleaved by factor D to form the bimolecular complex CVF,Bb. CVF,Bb is a C3/C5 convertase that cleaves both complement components C3 and C5. CVF is a three-chain protein that structurally resembles the C3b degradation product C3c, which is unable to form a C3/C5 convertase. Both C3 and CVF are synthesized as single-chain prepro-proteins. This study reports the recombinant expression of pro-CVF in two insect cell expression systems (baculovirus-infected Sf9 Spodoptera frugiperda cells and stably transfected S2 Drosophila melanogaster cells). In both expression systems pro-CVF is synthesized initially as a single-chain pro-CVF molecule that is subsequently proteolytically processed into a two-chain form of pro-CVF that structurally resembles C3. The C3-like form of pro-CVF can be further proteolytically processed into another two-chain form of pro-CVF that structurally resembles C3b. Unexpectedly, all three forms of pro-CVF exhibit functional activity of mature, natural CVF. Recombinant pro-CVF supports the activation of factor B in the presence of factor D and Mg2+ and depletes serum complement activity like natural CVF. The bimolecular convertase pro-CVF,Bb exhibits both C3 cleaving and C5 cleaving activity. The activity of pro-CVF and the resulting C3/C5 convertase is indistinguishable from CVF and the CVF,Bb convertase. The ability to produce active forms of pro-CVF recombinantly ensures the continued availability of an important research reagent for complement depletion because cobra venom as the source for natural CVF will be increasingly difficult to obtain as the Indian cobra is on the list of endangered species. Experimental systems to express pro-CVF recombinantly will also be invaluable for studies to delineate the structure and function relationship of CVF and its differences from C3 as well as to generate human C3 derivatives with CVF-like function for therapeutic complement depletion ("humanized CVF").     

Isolation and characterization of a 33,000-dalton fragment of complement Factor B with catalytic and C3b binding activity

Factor B is the zymogen of the catalytic site bearing subunit Bb of the C3/C5 convertase of the alternative pathway of complement. In this study, the location of the C3b binding site and the catalytic site within the Bb subunit were investigated. When human Factor B was treated with porcine elastase, fragments with respective molecular weights of 36,000, 35,000, 33,000, 31,000, and 25,000 were generated. Binding studies showed that only the 33,000-dalton fragment was capable of binding to C3b. The 33,000-dalton fragment was purified using fast protein liquid chromatography and found to be part of the Bb fragment upon testing with monoclonal antibody 15-6-19-1. Amino-terminal amino acid sequence analysis of the 33,000-dalton fragment placed it in the C-terminal half of Bb. The fragment expressed esterolytic activity as evidenced by cleavage of the synthetic substrate N alpha-acetyl-glycyl-L-lysine methyl ester and restored alternative pathway activity in Factor B-depleted serum. Its hemolytic activity was approximately 60-fold lower than that of Factor B. Comparative binding studies in the presence of metal ions using zymosan-C3b showed that the 33,000-dalton fragment bound to C3b with higher affinity than Factor B. Addition of the fragment to human serum inhibited alternative pathway activation by rabbit erythrocytes due to its high affinity for C3b and its low hemolytic activity compared to Factor B. These results show that the C-terminal 33,000-dalton portion of Bb contains not only the enzymatic site of Bb but also a C3b binding site which confers hemolytic activity upon the fragment. The observation that the fragment inhibited alternative pathway activation suggests that a synthetic peptide may be constructed that exhibits negative regulator activity in the alternative pathway.     

C5a induction of human interleukin 1. Synergistic effect with endotoxin or interferon-gamma

Interleukin-1 (IL-1) is a potent cytokine which possesses the ability to mediate systemic acute phase responses as well as local tissue inflammation. In these studies, we have examined the ability of C5a and C5a des Arg to induce IL-1 production in vitro. Human C5a and C5a des Arg were purified to homogeneity and were found to stimulate IL-1 release from freshly obtained human mononuclear cells into the extracellular medium. Only 2 hr of exposure to the purified complement components were necessary in order to stimulate IL-1 production. The minimal concentration of C5a required was 25 ng/ml, whereas 125 ng/ml of C5a des Arg induced comparable amounts of IL-1. This dose relationship was maintained at higher concentrations (150 ng/ml vs 750 ng/ml, respectively). That the effect was due to the anaphylatoxins themselves, and not endotoxin contamination, was shown by negative Limulus amebocyte lysate tests and employing preincubation of C5a/C5a des Arg with polymyxin B. The latter blocked a wide dose range of endotoxin-stimulated IL-1 production. However, when endotoxin was added to C5a or C5a des Arg, significant synergism in the stimulation of IL-1 production was observed, occurring at various concentrations of either agent. A similar synergism with C5a/C5a des Arg was seen with interferon-gamma. In these studies, IL-1 production was measured by bioassay employing cloned D . 10 . G4 . 1 murine T cells and by radioimmunoassay for human IL-1 beta; using C5a/C5a des Arg as stimulants, there was a high degree of correlation (r = 0.82) between the two assays. Since traumatic, infectious, and inflammatory diseases may result in the simultaneous appearance of these stimuli, the synergism described herein is likely to be clinically relevant.     

A direct in vivo comparison of the inflammatory properties of human C5a and C5a des Arg in human skin

C5a is an 11,000-Da complement-derived inflammatory glycoprotein that has been shown to mediate inflammatory reactions in vitro as well as in vivo in human skin. The C5a degradation product, C5a des Arg, is rapidly formed after exposure of C5a to serum carboxypeptidase N and may represent the relevant C5-derived inflammatory peptide in vivo. To examine the biologic activity of human C5a des Arg in vivo and to compare it with that seen with human C5a, we purified and characterized homogeneous preparations of human C5a and C5a des Arg and injected them intradermally into seven normal volunteers. C5a des Arg exhibited biochemical and biologic properties in vitro that were different from those of C5a. When injected into human skin, C5a des Arg was less potent than C5a, in respect to both minimal dose eliciting wheal and flare reactions and maximal wheal and flare elicited at a given dose, but C5a des Arg still elicited cutaneous wheal and flare reactions at physiologically relevant concentrations. Histologically, C5a des Arg skin test sites showed dense polymorphonuclear neutrophil-rich infiltrates associated with leukocytoclasis, dermal mast cell degranulation, and endothelial cell swelling. These were virtually indistinguishable from reactions elicited by C5a and occurred with concentrations attainable in vivo. Cutaneous wheal and flare reactions elicited by either C5a or C5a des Arg were partially inhibited by H1 antihistamines but were unaffected by selected nonsteroidal anti-inflammatory agents.     

Ascorbic acid transport by 3T6 fibroblasts. Regulation by and purification of human serum complement factor

It was earlier reported (Padh, H., and Aleo, J. J. (1987) Proc. Soc. Exp. Biol. Med. 185, 153-157) that the activation of serum complement by endotoxin or immunocomplexes inhibited ascorbate transport in 3T6 fibroblasts. We show here that the inhibitor of 3T6 fibroblasts. We show here that the inhibitor of ascorbate transport increased the Km for ascorbate without affecting the Vmax, indicating that the inhibitor reduces the affinity of the ascorbate transporter for ascorbate without affecting the process of translocation. Inhibition by serum and endotoxin was reversible, and the generated inhibitor was no longer heat-labile (at 56 degrees C for 30 min) suggesting that the inhibitor of ascorbate transport is likely to be a small protein molecule. Utilization of complement components suggested that C3 was consumed during formation of the inhibitor of ascorbate transport while C5 and factor B were not consumed. These data along with other results indicate that the inhibitor is generated at C3 step of complement activation. The inhibitor was purified from inulin activated human serum and it had an apparent molecular mass of around 9000 daltons. The inhibitory effect of the purified factor was abolished by antiserum to C3a suggesting that the 9000-dalton factor could be related to this fragment of complement protein. These data raise the possibility that tissue supply of ascorbate may be compromised during infection or autoimmune processes when serum complement is activated.     

Alternative complement pathway activation fragment Ba binds to C3b. Evidence that formation of the factor B-C3b complex involves two discrete points of contact

Alternative complement pathway C3 convertase formation involves the cleavage of C3b-associated factor B into fragments Ba and Bb. Whereas Bb, in complex with C3b, has proteolytic specificity toward native C3, the function of the Ba moiety in the formation and/or decay of alternative complement pathway C3 convertase is uncertain. Therefore, we have examined the effect of purified Ba fragment on both fluid-phase and surface-bound enzymatic activity and showed that whereas Ba could inhibit the rate of C3 convertase formation, the rate of intrinsic decay remained unaffected. A specific, metal ion-independent interaction between Ba and C3b was subsequently demonstrated by use of the cross-linking reagent dithiobis(succinimidyl propionate). When cell-associated 125I-B was activated by D, the dissociation of Bb fragment displayed simple first-order kinetics with a half-time of 2.4 min, this value being in reasonable agreement with the hemolytically determined decay rate of 1.8 min. In contrast, most of the Ba fragment undergoes rapid dissociation, but there is also evidence to suggest the establishment of a new equilibrium due to the ability of Ba to rebind to C3b. Cumulatively, these data are consistent with a model in which the attachment of intact B to C3b is mediated by two points of contact, one being in the Ba domain and the other in the Bb domain. Due to avidity effects, each of these interactions could be of relatively low intrinsic affinity, and the characteristic unidirectionality of alternative complement pathway C3 convertase decay may simply result from the low intrinsic association of "univalent" Bb for the C3b subunit.     

A new apoptotic pathway for the complement factor B-derived fragment Bb

Apoptosis is involved in both the cellular and humoral immune system destroying tumors. An apoptosis-inducing factor from HL-60 myeloid leukemia cells was obtained, purified, and sequenced. The protein found has been identified as a human complement factor B-derived fragment Bb, although it is known that factor B is able to induce apoptosis in several leukemia cell lines. Monoclonal antibodies against fragment Ba and Bb inhibited the apoptotic activity of factor B. When the purified fragment Bb was used for apoptosis induction, only the anti-Bb antibody inhibited Bb-induced apoptosis, and not the anti-Ba antibody. The apoptosis-inducing activity was found to be enhanced under conditions facilitating the formation of Bb. Blocking TNF/TNFR or FasL/Fas interactions did not interfere with the factor B-induced apoptosis. CD11c (iC3bR) acts as the main subunit of a heterodimer binding to fragment Bb in the apoptosis pathway, and the factor B-derived fragment Bb was found to possess the previously unknown function of inducing apoptosis in leukemic cells through a suicide mechanism of myeloid lineage cells during the differentiation stage.     

Chemotactic responses of human peripheral blood monocytes to the complement-derived peptides C5a and C5a des Arg

We examined responses of human peripheral blood polymorphonuclear leukocytes (PMN) and monocytes to the highly purified human complement-derived peptides C5a and C5a des Arg. As reported previously, C5a proved to be approximately 10- to 20-fold more potent than C5a des Arg as a chemoattractant for human PMN. C5a also was more potent than C5a des Arg in causing PMN to acquire a polarized morphology. In contrast, we found that human monocytes do not distinguish between C5a and C5a des Arg when these peptides are used as chemoattractants. In two different assay systems, both peptides acted at identical concentrations to stimulate suboptimal and optimal migration of monocytes. Human monocytes also did not distinguish between C5a and C5a des Arg when these peptides were used as inducers of polarization. Studies performed with functionally active, [125I]-labeled C5a and C5a des Arg, however, demonstrated that binding of C5a des Arg to monocytes differed from binding of C5a. Although [125I]-C5a des Arg appeared to bind to the same receptor as [125I]-C5a, binding of labeled C5a des Arg occurred with an affinity that was approximately 100-fold less than that observed with labeled C5a. These results indicate that leukocyte chemotactic and polarization responses to C5a and C5a des Arg vary, depending on the target cell type.     

Human complement proteins D, C2, and B. Active site mapping with peptide thioester substrates

The specificity and reactivity of complement serine proteases D, B, Bb, C2, and C2a were determined using a series of peptide thioester substrates. The rates of thioester hydrolysis were measured using assay mixtures containing the thiol reagent 4,4'-dithiodipyridine at pH 7.5. Each substrate contained a P1 arginine residue, and the effect of various groups and amino acids in the P2, P3, P4, and P5 positions was determined using kcat/Km values to compare reactivities. Among peptide thioesters corresponding to the activation site sequence in B, dipeptide thioesters containing a P2 lysine residue were the best substrates for D. Extending the chain to include a P3 or P4 amino acid resulted in loss of activity, and neither the tripeptide nor the tetrapeptide containing the cleavage sequence of B was hydrolyzed. Overall, D cleaved fewer substrates and was 2-3 orders of magnitude less reactive than C1s against some thioester substrates. C2 and fragment C2a had comparable reactivities and hydrolyzed peptides containing Leu-Ala-Arg and Leu-Gly-Arg, which have the same sequence as the cleavage sites of C3 and C5, respectively. The best substrates for C2 and C2a were Z-Gly-Leu-Ala-Arg-SBzl and Z-Leu-Gly-Leu-Ala-Arg-SBzl, respectively, where Bzl is benzyl. B was the least reactive among these complement enzymes. The best substrate for B was Z-Lys-Arg-SBzl with a kcat/Km value of 1370 M-1 s-1. The catalytic fragment of B, Bb, had higher activity toward these peptide thioester substrates. The best substrate for Bb was Z-Gly-Leu-Ala-Arg-SBzl with a kcat/Km similar to C2a and 10 times higher than the value for B. Both C2a and Bb were considerably more reactive against C3-like than C5-like substrates. Bovine trypsin hydrolyzed thioester substrates with kcat/Km approximately 10(3) higher than the complement enzymes. These thioester substrates for D, B, and C2 should be quite useful in kinetic and active site studies of the purified enzymes.     

Activation of the alternative complement pathway and generation of stimulating factors for granulocytes by glass fibers

Continuous-filament glass fibers coated with organic agents, candidate asbestos substitutes, were assessed for their ability to elicit from normal human serum complement-derived cleavage products which are able to stimulate the chemotaxis and the respiratory burst of polymorphonuclear leukocytes. Glass fibers generated chemoattracting and respiratory stimulating factors for polymorphonuclears from human serum. The effect was dose related for chemotaxis from the serum fiber concentration of 75 g/ml to 1,250 g/ml. The serum chemoattracting activity, as well the respiratory stimulation, were dramatically impaired when serum had been preliminarily absorbed with antiC5 antiserum. Since the impairment of chemotactic activity occurred also in the presence of EDTA, but not in the presence of EGTA, we assumed an activation of the alternative complement pathway.Glass fibers were studied in comparison to a UICC sample of Canadian chrysotile asbestos, which is able to activate in vitro the alternative complement pathway.Glass fibers exhibited less ability than asbestos fibers to generate complement cleavage products with chemotactic activity for polymorphonuclears; however, they produced an activity about equal to 80% of a chemotactic standard stimulus such as zymosan-activated plasma.Abbreviations AF asbestos fibers - antiCS-abs-S NHS absorbed with antiserum against C5 - EDTA-CH-S NHS treated with EDTA - EGTA-Ch-S NHS treated with EGTA - GF continuous filament glass fibers coated with a binder of organic substances - NHP normal human plasma - NHS normal human serum - PMN polymorphonuclear luekocytes - ZAP zymosan-activated plasma     

Stabilization of the amplification convertase of complement by monoclonal antibodies directed against human factor B

Three IgM mouse monoclonal antibodies (MoAb), 5B5-A, 2D2-B, and 5B12-A, were prepared by fusion of spleen cells from mice immunized with human B with the SP 2/0 myeloma cell line. They were assessed for their effect on cell-bound and fluid-phase amplification convertases of complement (C) with purified proteins in vitro. 5B5-A and 2D2-B were similar in their effects on cell-bound preformed C3bBb in that they bound to cell-bound C3bBb, stabilized the C3bBb convertase, and rendered the C3bBb convertase relatively resistant to the plasma protein H. These two MoAb were also able to enhance C3 consumption in vitro in reaction mixtures containing C3b, C3, B, and D. At the same time, they presumably stabilized the C3 convertase and caused relative sparing of B hemolytic activity in the reaction mixtures. In contrast, 5B12-A, which also bound to Bb and C3bBb, did not induce any stabilization, but rather caused accelerated decay of cell-bound C3bBb. These results indicate that MoAb against B can have C3NeF-like activity. On the other hand, not all MoAb against B have stabilizing activity on the C3bBb convertase.