Experimental design of complement component 5a-induced acute lung injury (C5a-ALI): a role of CC-chemokine receptor type 5 during immune activation by anaphylatoxin.

Excessive activation of the complement system is detrimental in acute inflammatory disorders. In this study, we analyzed the role of complement-derived anaphylatoxins in the pathogenesis of experimental acute lung injury/acute respiratory distress syndrome (ALI/ARDS) in C57BL/6J mice. Intratracheal administration of recombinant mouse complement component (C5a) caused alveolar inflammation with abundant recruitment of Ly6-G(+)CD11b(+) leukocytes to the alveolar spaces and severe alveolar-capillary barrier dysfunction (C5a-ALI; EC(50[C5a]) = 20 ng/g body weight). Equimolar concentrations of C3a or desarginated C5a (C5a(desArg)) did not induce alveolar inflammation. The severity of C5a-ALI was aggravated in C5-deficient mice. Depletion of Ly6-G(+) cells and use of C5aR1(-/-) bone marrow chimeras suggested an essential role of C5aR1(+) hematopoietic cells in C5a-ALI. Blockade of PI3K/Akt and MEK1/2 kinase pathways completely abrogated lung injury. The mechanistic description is that C5a altered the alveolar cytokine milieu and caused significant release of CC-chemokines. Mice with genetic deficiency of CC-chemokine receptor (CCR) type 5, the common receptor of chemokine (C-C motif) ligand (CCL) 3, CCL4, and CCL5, displayed reduced lung damage. Moreover, treatment with a CCR5 antagonist, maraviroc, was protective against C5a-ALI. In summary, our results suggest that the detrimental effects of C5a in this model are partly mediated through CCR5 activation downstream of C5aR1, which may be evaluated for potential therapeutic exploitation in ALI/ARDS.

Structural basis for the targeting of complement anaphylatoxin C5a using a mixed L-RNA/L-DNA aptamer.

L-Oligonucleotide aptamers (Spiegelmers) consist of non-natural L-configured nucleotides and are of particular therapeutic interest due to their high resistance to plasma nucleases. The anaphylatoxin C5a, a potent inflammatory mediator generated during complement activation that has been implicated with organ damage, can be efficiently targeted by Spiegelmers. Here, we present the first crystallographic structures of an active Spiegelmer, NOX-D20, bound to its physiological targets, mouse C5a and C5a-desArg. The structures reveal a complex 3D architecture for the L-aptamer that wraps around C5a, including an intramolecular G-quadruplex stabilized by a central Ca(2+) ion. Functional validation of the observed L-aptamer:C5a binding mode through mutational studies also rationalizes the specificity of NOX-D20 for mouse and human C5a against macaque and rat C5a. Finally, our structural model provides the molecular basis for the Spiegelmer affinity improvement through positional L-ribonucleotide to L-deoxyribonucleotide exchanges and for its inhibition of the C5a:C5aR interaction.

Decreased survival of human breast cancer cells expressing HER2/neu on in vitro incubation with an anti-HER2/neu antibody fused to C5a or C5a desArg.

Treatment of human epidermal growth factor receptor 2 (HER2/neu)-expressing breast cancer patients with a monoclonal antibody (mAb) directed against HER2/neu improves the outcome of chemotherapy. In cases in which remission is observed, antibody-dependent cell-mediated cytotoxicity (ADCC) seems to be one of the main mechanisms of anti-HER2/neu mAb action, implicating Fc gamma receptors (Fc gamma Rs) in this tumoricidal activity. In vitro and in vivo studies have revealed that anti-HER2/neu-mediated ADCC is mainly accomplished by polymorphonuclear granulocytes (PMN). C5a, a cleavage product of the complement component C5, modulates Fc gamma R expression via upregulation of activating and downregulation of inhibitory Fc gamma Rs. C5a also recruits PMNs to sites of inflammation and increases PMN survival. To enhance the recruitment and activation of C5a receptor-bearing cells into the tumor microenvironment, we developed antibody fusion proteins composed of a human IgG3 anti-HER2/neu antibody genetically fused to C5a [anti-HER2/neu IgG3-(C5a)] or to its derivative, C5a(desArg) [anti-HER2/neu IgG3-(C5a(desArg))]. Both fusion proteins were expressed, properly assembled, and secreted by murine myeloma cells, and displayed chemotactic activity on human PMN. Under comparable conditions, anti-HER2/neu IgG3-(C5a(desArg)) increased the survival of PMN more efficiently than anti-HER2/neu IgG3-(C5a) or C5a(desArg). Surprisingly, incubation of the fusion proteins with breast cancer cells that overexpress HER2/neu (SK-BR-3) induced cell death at a dose at which the anti-HER2/neu IgG3 antibody was innocuous. In the presence of human peripheral blood leukocytes as effector cells, both fusion proteins induced tumor cell death more efficiently than anti-HER2/neu IgG3. These data suggest that anti-HER2/neu IgG3-(C5a) and anti-HER2/neu IgG3-(C5a(desArg)) fusion proteins possess novel properties that could be useful in cancer immunotherapy.

Correlation of plasma complement split product levels with allergic respiratory disease activity and relation to allergen immunotherapy.

BACKGROUND: Allergens, including dust mite and grass pollen, and mast cell tryptase are known to generate the complement split products (CSPs) C5a and C3a, which can then trigger allergic inflammation. The relation of these anaphylatoxin levels to clinical allergic disease responses is not known. OBJECTIVE: To evaluate the relationship of plasma CSP levels to allergic respiratory disease variables in an adult cohort. METHODS: A cross-sectional survey was used to assess the association of plasma C5a desArg and C3a desArg levels with clinical allergic respiratory disease variables. Furthermore, a time course of the effect of routine allergen immunotherapy on plasma CSP levels and cutaneous and pulmonary responses was determined. RESULTS: Adult plasma C5a desArg levels correlate with asthma severity as determined by a physician (P = .01) and by Asthma Quality of Life Questionnaire scores (P < .01). Change in plasma C5a desArg levels 1 hour after immunotherapy is associated with baseline rhinoconjunctivitis symptom severity (P = .03), change in total mean wheal diameter (P = .05), and total dust mite dosage (P = .04). Change in plasma C3a desArg levels 3 hours after immunotherapy correlates with change in total mean wheal diameter induced by dust mite (P = .01). Change in plasma CSP levels after immunotherapy did not correlate with change in spirometric outcome. CONCLUSIONS: Plasma C5a desArg levels reflect allergic respiratory disease severity as assessed by physicians and correlate with Asthma Quality of Life Questionnaire scores. Changes in CSP levels after immunotherapy reflect cutaneous allergic responses, especially to dust mite allergen.

Structure of human desArg-C5a.

The anaphylatoxin C5a is derived from the complement component C5 during activation of the complement cascade. It is an important component in the pathogenesis of a number of inflammatory diseases. NMR structures of human and porcine C5a have been reported; these revealed a four-helix bundle stabilized by three disulfide bonds. The crystal structure of human desArg-C5a has now been determined in two crystal forms. Surprisingly, the protein crystallizes as a dimer and each monomer in the dimer has a three-helix core instead of the four-helix bundle noted in the NMR structure determinations. Furthermore, the N-terminal helices of the two monomers occupy different positions relative to the three-helix core and are completely different from the NMR structures. The physiological significance of these structural differences is unknown.

Systemic hypoxia enhances exercise-mediated bactericidal and subsequent apoptotic responses in human neutrophils.

Phagocytosis and oxidative burst are critical host defense mechanisms in which neutrophils clear invading pathogens. Clearing phagocytic neutrophils by triggering apoptosis is an essential process for controlling inflammation. This study elucidates how various exercise bouts with/without hypoxia affected neutrophil bactericidal activity and subsequent apoptosis in humans. Fifteen sedentary males performed six distinct experimental tests in an air-conditioned normobaric hypoxia chamber: two normoxic exercises [strenuous exercise (SE; up to maximal O2 consumption) and moderate exercise (ME; 50% maximal O2 consumption for 30 min) while exposed to 21% O2], two hypoxic exercises (ME for 30 min while exposed to 12% and 15% O2), and two hypoxic exposures (resting for 30 min while exposed to 12% and 15% O2). The results showed that 1) plasma complement-C3a desArg/C4a desArg/C5a concentrations were increased, 2) expressions of L-selectin/lymphocyte functin-associated antigen-1/Mac-1/C5aR on neutrophils were enhanced, 3) phagocytosis of neutrophils to Esherichia coli and release of neutrophil oxidant products by E. coli were elevated, and 4) E. coli-induced phosphotidylserine exposure or caspase-3 activation of neutrophils were promoted immediately and 2 h after both 12% O2 exposure at rest and with ME as well as normoxic SE. Although neither normoxic ME nor breathing 15% O2 at rest influenced these complement- and neutrophil-related immune responses, ME at both 12% and 15% O2 resulted in enhanced complement activation in the blood, expressions of opsonic/complement receptors on neutrophils, or the bactericidal activity and apoptosis of neutrophils. Moreover, the increased neutrophil oxidant production and apoptosis by normoxic SE and hypoxic ME were ameliorated by treating neutrophils with diphenylene iodonium (a NADPH oxidase inhibitor). Therefore, we conclude that ME at 12-15% O2 enhances bactericidal capacity and facilitates the subsequent apoptosis of neutrophils.

Functions of C5a receptors.

The split product of the complement protein, C5, is C5a and is an extremely potent pro-inflammatory peptide that interacts with two C5a receptors, C5aR and C5L2, present on surfaces of phagocytes as well as other cell types. The former is a well-established receptor that initiates G-protein-coupled signaling via mitogen-activated protein kinase pathways. Its in vivo blockade greatly reduces inflammatory injury. Much less is known about C5L2, occupancy of which by C5a does not initiate increased intracellular Ca(2+). There are numerous conflicting reports suggesting that C5L2 is a "default receptor" that attenuates C5a-dependent biological responses by competing with C5aR for binding of C5a. However, there are other reports suggesting that C5L2 plays an active, positive role in inflammatory responses. Better definition of C5L2 is needed if its in vivo blockade, along with C5aR, is to be considered in complement-dependent inflammatory diseases.

The role of the N-terminal domain of the complement fragment receptor C5L2 in ligand binding.

C5L2 is a new cellular receptor found to interact with the human anaphylatoxins complement factor C5a and its C-terminal cleavage product C5a des Arg. The classical human C5a receptor (C5aR) preferentially binds C5a, with a 10-100-fold lower affinity for C5a des Arg. In contrast, C5L2 binds both ligands with nearly equal affinity. C5aR presents acidic and tyrosine residues in its N terminus that interact with the core of C5a while a hydrophobic pocket formed by the transmembrane helices interacts with residues in the C terminus of C5a. Here, we have investigated the molecular basis for the increased affinity of C5L2 for C5a des Arg. Rat and mouse C5L2 preferentially bound C5a des Arg, whereas rodent C5aR showed much higher affinity for intact C5a. Effective peptidic and non-peptidic ligands for the transmembrane hydrophobic pocket of C5aR were poor inhibitors of ligand binding to C5L2. An antibody raised against the N terminus of human C5L2 did not affect the binding of C5a to C5L2 but did inhibit C5a des Arg binding. A chimeric C5L2, containing the N terminus of C5aR, had little effect on the affinity for C5a des Arg. Mutation of acidic and tyrosine residues in the N terminus of human C5L2 revealed that 3 residues were critical for C5a des Arg binding but had little involvement in C5a binding. C5L2 thus appears to bind C5a and C5a des Arg by different mechanisms, and, unlike C5aR, C5L2 uses critical residues in its N-terminal domain for binding only to C5a des Arg.

A functional C5a anaphylatoxin receptor in a teleost species.

The anaphylatoxins are potent, complement-derived low m.w. proteins that bind to specific seven-transmembrane receptors to elicit and amplify a variety of inflammatory reactions. C5a is the most potent of these phlogistic peptides and is a strong chemoattractant for neutrophils and macrophages/monocytes. Although lower vertebrates possess complement systems that are believed to function similarly to those of mammals, anaphylatoxin receptors have not previously been characterized in any nonmammalian vertebrate. To study the functions of C5a in teleost fish, we generated recombinant C5a of the rainbow trout, Oncorhynchus mykiss (tC5a), and used fluoresceinated tC5a (tC5aF) and flow cytometry to identify the C5a receptor (C5aR) on trout leukocytes. Granulocytes/Macrophages present in cell suspensions of the head kidney (HKL), the main hemopoietic organ in teleosts, showed a univariate type of receptor expression, whereas those from the peripheral blood demonstrated either a low or high level of expression. The binding of tC5aF was inhibited by excess amounts of unlabeled tC5a or tC5a(desArg), demonstrating that sites other than the C-terminal of tC5a interact with the C5aR. Both tC5a and tC5a(desArg) were able to induce chemotactic responses in granulocytes in a concentration-dependent manner, but the desArg derivative was at least 10-fold less active. Homologous desensitization occurred after HKL were exposed to continuous or high concentrations of tC5a, with a loss of tC5aF binding and an 80% reduction in chemotactic responses toward tC5a. Pertussis toxin reduced the migration of HKL toward tC5a by 40%, suggesting only a partial involvement of pertussis toxin-sensitive G(i) proteins in tC5a-mediated chemotaxis.

C5L2, a nonsignaling C5A binding protein.

C5a anaphylatoxin, a potent inflammatory mediator, is known to act through a specific G protein coupled receptor. However, some of the complex effects of C5a in vivo may not be explained solely by the deletion of the known receptor. Here, we show that an orphan receptor, identified as C5L2, is a high affinity C5a binding protein. Unlike the previously described C5aR, C5L2 is obligately uncoupled from heterotrimeric G proteins, in part by virtue of an amino acid alteration in the so-called DRY sequence at the end of the third transmembrane segment. Both human and murine C5L2 bear a leucine for arginine replacement at this site. C5L2, when transfected into several cell types, is weakly phosphorylated in transfected cells following binding of C5a but does not induce significant activation of MAP kinases, mediate calcium flux, or stimulate chemotaxis. Bone marrow cells from wild type respond robustly to C5a with induction and suppression of a number of inflammation related genes. In contrast, C5a receptor deficient mice, which bear C5L2 alone, do not respond to C5a with changes in gene transcription by microarray analyses. Biophysical properties of the C5L2, including slow ligand on and off rates, absence of internalization, and relatively high affinity for the C5a des Arg metabolite, suggest that this receptor may serve to modulate C5a biological functions in vivo. Finally, in contrast to previous reports, we find absolutely no interaction of C5L2 with other anaphylatoxins C3a and C4a.

Requirements for C5a receptor-mediated IL-4 and IL-13 production and leukotriene C4 generation in human basophils.

Anaphylatoxin derived from the fifth complement component (C5a) in the presence of IL-3 induces continuous leukotriene C4 generation and IL-4 and IL-13 expression in human basophils for a period of 16-18 h. This indicates that the G protein-coupled C5a receptor (C5aR) can induce long-lasting cellular responses. Using anti-N-terminal C5aR Abs, C-terminal C5a hexapeptide analogs, and pertussis toxin, we demonstrate that the putative activation site of the C5aR is both necessary and sufficient for these late cellular responses. Furthermore, continuous pertussis toxin-sensitive G protein-coupled receptor activation and receptor-ligand interaction is ongoing and required during the entire period of product release. However, the late basophil responses have a more stringent requirement for optimal receptor activation. Leukotriene C4 generation appears to be influenced mostly by the way the receptor is activated, because the most active hexapeptide is a superagonist for this response. By contrast, C5adesarg, lacking the C-terminal arginine, induces minimal lipid mediator formation but is fully active to induce IL-4 production and is even a superagonist for IL-13 release. Nevertheless, IL-4/IL-13 synthesis in response to C5adesarg could be blocked by both C-terminal antagonistic peptide as well as anti-N-terminal C5aR Abs, indicating only minor differences of ligand-receptor interactions between C5a and C5adesarg. Taken together, our data demonstrate that long-lasting and continuous signaling occurs through a limited activation domain of the C5aR, which can differentially promote separate basophil functions.

Receptor activation by human C5a des Arg74 but not intact C5a is dependent on an interaction between Glu199 of the receptor and Lys68 of the ligand.

Despite the expression of only one type of receptor, there is great variation in the ability of different cell types to discriminate between C5a and its more stable metabolite, C5a des Arg74. The mechanism that underlies this phenomenon is not understood but presumably involves differences in the interaction with the C5a receptor. In this paper, we have analyzed the effects of a substitution mutation of the receptor (Glu199 --> Lys199) and the corresponding reciprocal mutants (Lys68 --> Glu68) of C5a, C5a des Arg74 and peptide analogues of the C-terminus of C5a on the ability of the C5a receptor to discriminate between ligands with and without Arg74. The use of these mutants indicates that the Lys68/Glu199 interaction is essential for activation of receptor by C5a des Arg74 but not for activation by intact C5a. The substitution of Asp for Arg74 of C5a [Lys68] produces a ligand with equal potency on both the wild-type and mutant receptors, suggesting that it is the C-terminal carboxyl group rather than the side chain of Arg74 that controls the responsiveness of the receptor to Lys68. In contrast, the mutation of Lys68 to Glu(68) has little effect on the ability of either C5a or C5a des Arg(74) to displace [(125)I]C5a from the receptors, indicating that binding of ligand and receptor activation are distinct but interdependent events. C5a and the truncated ligand, C5a des Arg74, appear to have different modes of interaction with the receptor and the ability of the human C5a receptor to discriminate between these ligands is at least partly dependent on an interaction with the receptor residue, Glu199.

Analysis of the C5a anaphylatoxin core domain using a C5a phage library selected on differentiated U937 cells.

The human anaphylatoxin C5a is a 74-amino acid comprising polypeptide with a plethora of biological functions. Site directed mutagenesis studies suggest that several residues within the core and the C-terminus mediate the interaction with the C5a receptor. However, the contribution of particular core residues to receptor binding remained to be clarified. By means of the phage display technique, the loop between positions 35-40 was randomly mutated and the resulting C5a[35-40] fusion phage library affinity selected on C5a receptor expressing U937 cells. After five rounds of affinity enrichment, residues Arg37 and Arg40 were preferably selected. Enrichment was as high as 100% for Arg37 and 79% for Arg40. No significant enrichment of consensus residues could be obtained for positions 35, 36, 38 and 39. The core mutant C5a[A35E36R37A38S39R40], in which only Arg37/40 and Ala38 are of the native C5a sequence, was as potent as native C5a in both receptor binding and enzyme release examined on U937 cells. In contrast, replacement of Arg40 as in the mutant C5a[Q35E36R37I38L39N40] resulted in a 10-fold decrease in both binding and functional activities. Thus, selected out of a multiplicity of possibilities by the natural binding partner, Arg37 as well as Arg40 appear to be anchor residues in binding to the C5a receptor.

Identification of neutrophil chemotactic factors in bronchoalveolar lavage fluid of asthmatic patients.

BACKGROUND: Although neutrophils have been implicated in bronchial asthma, the mechanism(s) which bring these cells into the airways is poorly understood. OBJECTIVE: To investigate the presence and identity of neutrophil chemotactic factors in bronchoalveolar lavage (BAL) fluid from atopic asthmatic subjects. METHOD: BAL fluid was obtained from 13 subjects (seven asthmatics and six normals), aged 19 to 60 yr, at bronchoscopy. Separation of neutrophil chemotactic activity (NCA) was achieved by FPLC cation exchange chromatography. Fractions were collected and assayed for chemotaxis in multiwell micro-chemotaxes chambers using polycarbonate filters, for the complement peptide C5a/C5a des Arg by radioimmunoassay (RIA) and for interleukin-8 (IL-8) by ELISA. RESULTS: NCA was found in FPLC fractions of BAL samples in four out of seven asthmatics and each of these subjects had at least three similar peaks of NCA. The major peak of NCA was found to contain immunoreactive C5a/C5a des Arg and chemotaxis. In response to this NCA could be blocked by desensitization of the neutrophils with recombinant C5a. Purified serum derived C5a/C5a des Arg was found to have altered chromatographic properties when added to BAL fluid; this suggested that BAL fluid contained proteins which interacted with the C5a/C5a des Arg. Immunoreactive IL-8 (iIL-8) was also detected but its concentration or chemical form was insufficient to induce neutrophil chemotaxis. CONCLUSION: This study demonstrates that bronchial asthmatic lavage fluid contains C5a/ C5a des/Arg and IL-8, together with other as yet unidentified factors which may contribute to neutrophil recruitment in this disease.

Hemofiltration in human sepsis: evidence for elimination of immunomodulatory substances.

Continuous hemofiltration is widely used for renal replacement therapy in patients with acute renal failure. It has been suggested that hemofiltration may also eliminate toxic mediators thought to be important in the pathophysiology of sepsis. The present study examined whether hemofiltration can activate or eliminate inflammatory mediators in patients with sepsis, and whether ultrafiltrate can alter specific functions of peripheral blood mononuclear leukocytes (PBMC) in vitro. Veno-venous hemofiltration was performed in 16 patients and in 5 healthy volunteers. Pre-filter (afferent), post-filter (efferent) and ultrafiltrate concentrations of cytokines (IL-1 beta, IL-6, IL-8, TNF alpha) and of complement components (C3, C3adesArg, C5adesArg, terminal complement complex) were measured after the beginning of hemofiltration (t0), and 60 minutes later (t60). PBMC, and monocyte and lymphocyte subfractions were incubated with ultrafiltrate, and cytokines were determined in the supernatants. Hemofiltration did not induce significant mediator activation. There was no evidence for significant cytokine elimination. However, pre-filter C3adesArg concentration showed a significant decline during hemofiltration (patients: t0 = 676.9 +/- 99.7 ng/ml, t60 = 545.4 +/- 83.2, P < 0.001; volunteers: t0 = 54.8 +/- 13.3 ng/ml, t60 = 33.9 +/- 10.7, P < 0.001). Ultrafiltrate from septic patients significantly stimulated PBMC and monocyte TNF alpha release, but suppressed lymphocyte IL-2 and IL-6 production. Ultrafiltrate from volunteers was without effect. Hemofiltration effectively eliminates certain mediators such as C3adesArg. Ultrafiltrate contains compounds with significant immunomodulatory qualities. Therefore, hemofiltration may represent a new modality for removal of immunomodulatory mediators.

Binding of Gc globulin (vitamin D binding protein) to C5a or C5a des Arg is not necessary for co-chemotactic activity.

Gc globulin (vitamin D binding protein) has been shown to augment significantly the leukocyte chemotactic activity of the activated complement peptides C5a and C5a des Arg. However, the mechanism of chemotaxis enhancement is not known. Natural C5-derived peptides contain a carbohydrate side chain that comprises approximately 25% of the mass of the 11-kDa peptides. Previous studies have demonstrated that Gc globulin binds to C5-derived peptides via sialic acid residues on this carbohydrate side chain. The necessity of this carbohydrate side chain for chemotaxis enhancement by Gc globulin was investigated by using both natural (glycosylated) and recombinant (carbohydrate-free) peptides. The dose-response curves of neutrophil chemotaxis to recombinant C5a or C5a des Arg plus Gc globulin were identical to those observed with the naturally derived peptides, despite the fact that natural C5a bound to Gc globulin while the recombinant C5a failed to bind this protein. Moreover, neutrophils pretreated with Gc globulin then washed before addition to the chemotaxis assay displayed significantly enhanced movement to C5a alone. These results indicate that the binding of C5a/C5a des Arg to Gc globulin is not necessary for their chemotactic activity to be augmented. Furthermore, these results demonstrate that the co-chemotactic activity of Gc globulin is generated on the cell surface, independent of C5a binding to its receptor.

Inactivation of human anaphylatoxin C5a and C5a des-Arg through cleavage by the plasminogen activator activity of a human fibrosarcoma cell line.

The HT-1080 human fibrosarcoma cell line exhibited a plasminogen-dependent ability to inactivate recombinant anaphylatoxin C5a or zymosan-activated serum. The inactivation was obtained at physiological levels of both plasminogen (2 microM) and C5a (1-5 nM). Inactivated C5a and zymosan-activated serum were no longer able to induce chemotaxis and degranulation of neutrophils. Inactivation of C5a paralleled the emergence of plasmin activity, assayed by cleavage of the synthetic substrate H-D-valyl-L-leucyl-L-lysine-p-nitroanilide (S-2251). Both C5a inactivation and S-2251 cleavage were inhibited by the plasmin inhibitor alpha 2-antiplasmin, the urokinase inhibitor amiloride, and by anti-urokinase antibodies. In a cell-free system, inactivation of C5a was shown to depend on the simultaneous presence of urokinase and plasminogen and was inhibited by alpha 2-antiplasmin and by anti-urokinase antibodies. SDS-polyacrylamide electrophoresis demonstrated the cleavage of C5a by the plasminogen activation system and inhibition of the cleavage by amiloride. Amino acid sequencing of the band corresponding to the C5a degradation product revealed that C5a was cleaved at positions Lys14-His15 and Arg40-Ile41; cleavage at position Arg40-Ile41 seemed to be responsible for the loss of activity. Since neoplastic cells extensively produce and exhibit plasminogen activator activity, the present observations suggest that plasminogen activation may, by inactivation of C5a, reduce the anti-tumor immune response and support the immunological escape phenomenon of tumors.

Plasma clearance of the human C5a anaphylatoxin by binding to leucocyte C5a receptors.

The C5a anaphylatoxin is a potent complement-derived mediator of inflammation with chemotactic activity. In this study the possible role of specific high-affinity binding sites for C5a on peripheral blood leucocytes for the removal of C5a from human blood plasma was investigated. The addition of purified granulocytes or mononuclear cells to complement-activated plasma resulted in the rapid and dose-dependent removal of up to 80% of plasma C5a, as determined by ELISA. The specific role of leucocyte C5a receptors (C5aR) in the plasma clearance of C5a was demonstrated by the inhibition of C5a uptake by the preincubation of cells with the C5aR-specific monoclonal antibody S5/1. Furthermore, U937 cells which had been induced by db-cAMP to express C5aR, but not undifferentiated U937 cells, were capable of removing C5a from plasma. The inhibition of C5aR internalization by monensin did not affect C5a uptake by leucocytes. The co-incubation with leucocytes had no effect on the plasma clearance of complement activation products C3a or terminal complement complex (TCC), as determined by this in vitro assay. The binding of the C5a anaphylatoxin to cellular receptors represents an effective control mechanism that protects the organism from systemic effects of this potent phlogistic mediator.

Activation of the complement system and adverse effects of biodegradable pins of polylactic acid (Biofix) in osteochondritis dissecans.

Biodegradable pins of polyglycolic acid (PGA) or polylactic acid (PLA) have been used in the treatment of fractures and osteotomies during the past 5 years. Adverse effects reported have included swelling at the implantation site and sinus formation, considered to represent nonspecific foreign-body reactions. Recent reports, however, have shown severe reactions after intraarticular fracture fixation. Reactions in 2 patients, treated with polylactic pins for osteochondritis dissecans (OCD) in our hospital, prompted the present clinical investigation and further evaluation of the complement-activating potential of polylactic pins. 10 knees underwent arthroscopic fixation of an OCD-lesion with Biofix (PLA) pins. Clinical follow-ups were carried out at 2, 6, and 12 weeks and at 6 and 12 months. Blood samples were collected from 5 patients 9-24 months postoperatively for biocompatibility tests. Quantification of human C5a des Arg was performed with a recently developed sandwich ELISA technique, using neoepitope-specific monoclonal antibodies. 6 knees developed diffuse swelling and a prolonged postoperative course. 2 patients had a particularly prolonged course which could not be attributed to infection. Levels of C5a des Arg in plasma incubated in the presence of polylactic acid were higher than in plasma incubated in the absence of PLA. The high frequency of long-term postoperative inflammatory signs in these knees treated for OCD and the demonstration of a complement activation potential of PLA pins warrant further studies on the biocompatibility of this material. Until more information is available, we do not recommend intraarticular use of PLA pins.