Monocyte CD14 and HLA-DR expression increases with disease duration and severity in amyotrophic lateral sclerosis.

Objective: To investigate changes in immune markers and frequencies throughout disease progression in patients with amyotrophic lateral sclerosis (ALS). Methods: In this longitudinal study, serial blood samples were collected from 21 patients with ALS over a time period of up to 16 months. Flow cytometry was used to quantitate CD14, HLA-DR, and CD16 marker expression on monocyte subpopulations and neutrophils, as well as their cell population frequencies. A Generalized Estimating Equation model was used to assess the association between changes in these immune parameters and disease duration and the Revised Amyotrophic Lateral Sclerosis Functional Rating Scale (ALSFRS-R). Results: CD14 expression on monocyte subpopulations increased with both disease duration and a decrease in ALSFRS-R score in patients with ALS. HLA-DR expression on monocyte subpopulations also increased with disease severity and/or duration. The expression of CD16 did not change relative to disease duration or ALSFRS-R. Finally, patients had a reduction in non-classical monocytes and an increase in the classical to non-classical monocyte ratio throughout disease duration. Conclusion: The progressive immunological changes observed in this study provide further support that monocytes are implicated in ALS pathology. Monocytic CD14 and HLA-DR surface proteins may serve as a therapeutic target or criteria for the recruitment of patients with ALS into clinical trials for immunomodulatory therapies.

Phagocytosis of live and dead Escherichia coli and Staphylococcus aureus in human whole blood is markedly reduced by combined inhibition of C5aR1 and CD14.

BACKGROUND: Sepsis is a dysregulated host response to infection. The aim of this study was to investigate the effects of complement- and CD14 inhibition on phagocytosis of live and dead Gram-negative and Gram-positive bacteria in human whole blood. METHODS: Lepirudin-anticoagulated blood was incubated with live or dead E. coli or S. aureus at 37 °C for 120 min with or without the C5aR1 antagonist PMX53 and/or anti-CD14. Granulocyte and monocyte phagocytosis were measured by flow cytometry, and five plasma cytokines by multiplex, yielding a total of 28 mediators of inflammation tested for. RESULTS: 16/28 conditions were reduced by PMX53, 7/28 by anti-CD14, and 24/28 by combined PMX53 and CD14 inhibition. The effect of complement inhibition was quantitatively more pronounced, in particular for the responses to S. aureus. The effect of anti-CD14 was modest, except for a marked reduction in INF-ß. The responses to live and dead S. aureus were equally inhibited, whereas the responses to live E. coli were inhibited less than those to dead E. coli. CONCLUSION: C5aR1 inhibited phagocytosis-induced inflammation by live and dead E. coli and S. aureus. CD14 blockade potentiated the effect of C5aR1 blockade, thus attenuating inflammation.

Complement component 5 does not interfere with physiological hemostasis but is essential for Escherichia coli-induced coagulation accompanied by Toll-like receptor 4.

There is a close cross-talk between complement, Toll-like receptors (TLRs) and coagulation. The role of the central complement component 5 (C5) in physiological and pathophysiological hemostasis has not, however, been fully elucidated. This study examined the effects of C5 in normal hemostasis and in Escherichia coli-induced coagulation and tissue factor (TF) up-regulation. Fresh whole blood obtained from six healthy donors and one C5-deficient individual (C5D) was anti-coagulated with the thrombin inhibitor lepirudin. Blood was incubated with or without E. coli in the presence of the C5 inhibitor eculizumab, a blocking anti-CD14 monoclonal antibody (anti-CD14) or the TLR-4 inhibitor eritoran. C5D blood was reconstituted with purified human C5. TF mRNA was measured by quantitative polymerase chain reaction (qPCR) and monocyte TF and CD11b surface expression by flow cytometry. Prothrombin fragment 1+2 (PTF1·2) in plasma and microparticles exposing TF (TF-MP) was measured by enzyme-linked immunosorbent assay (ELISA). Coagulation kinetics were analyzed by rotational thromboelastometry and platelet function by PFA-200. Normal blood with eculizumab as well as C5D blood with or without reconstitution with C5 displayed completely normal biochemical hemostatic patterns. In contrast, E. coli-induced TF mRNA and TF-MP were significantly reduced by C5 inhibition. C5 inhibition combined with anti-CD14 or eritoran completely inhibited the E. coli-induced monocyte TF, TF-MP and plasma PTF1·2. Addition of C5a alone did not induce TF expression on monocytes. In conclusion, C5 showed no impact on physiological hemostasis, but substantially contributed to E. coli-induced procoagulant events, which were abolished by the combined inhibition of C5 and CD14 or TLR-4.

Staphylococcus aureus-induced complement activation promotes tissue factor-mediated coagulation.

Essentials Complement, Toll-like receptors and coagulation cross-talk in the process of thromboinflammation. This is explored in a unique human whole-blood model of S. aureus bacteremia. Coagulation is here shown as a downstream event of C5a-induced tissue factor (TF) production. Combined inhibition of C5 and CD14 efficiently attenuated TF and coagulation. SUMMARY: Background There is extensive cross-talk between the complement system, the Toll-like receptors (TLRs), and hemostasis. Consumptive coagulopathy is a hallmark of sepsis, and is often mediated through increased tissue factor (TF) expression. Objectives To study the relative roles of complement, TLRs and TF in Staphylococcus aureus-induced coagulation. Methods Lepirudin-anticoagulated human whole blood was incubated with the three S. aureus strains Cowan, Wood, and Newman. C3 was inhibited with compstatin, C5 with eculizumab, C5a receptor 1 (C5aR1) and activated factor XII with peptide inhibitors, CD14, TLR2 and TF with neutralizing antibodies, and TLR4 with eritoran. Complement activation was measured by ELISA. Coagulation was measured according to prothrombin fragment 1 + 2 (PTF1 + 2 ) determined with ELISA, and TF mRNA, monocyte surface expression and functional activity were measured with quantitative PCR, flow cytometry, and ELISA, respectively. Results All three strains generated substantial and statistically significant amounts of C5a, terminal complement complex, PTF1 + 2 , and TF mRNA, and showed substantial TF surface expression on monocytes and TF functional activity. Inhibition of C5 cleavage most efficiently and significantly inhibited all six markers in strains Cowan and Wood, and five markers in Newman. The effect of complement inhibition was shown to be completely dependent on C5aR1. The C5 blocking effect was equally potentiated when combined with blocking of CD14 or TLR2, but not TLR4. TF blocking significantly reduced PTF1 + 2 levels to baseline levels. Conclusions S. aureus-induced coagulation in human whole blood was mainly attributable to C5a-induced mRNA upregulation, monocyte TF expression, and plasma TF activity, thus underscoring complement as a key player in S. aureus-induced coagulation.

Properdin deficiency protects from 5-fluorouracil-induced small intestinal mucositis in a complement activation-independent, interleukin-10-dependent mechanism.

Intestinal mucositis is a serious complication of chemotherapy that leads to significant morbidity that may require dose or drug adjustments. Specific mitigating strategies for mucositis are unavailable, due partly to an incomplete understanding of the pathogenic mechanisms. We have previously shown an effect of properdin, a positive regulator of complement activation, in models of colitis. Here we use properdin-deficient (PKO ) mice to interrogate the role of properdin and complement in small intestinal mucositis. Mucositis was induced by five daily injections of 5-fluorouracil (5-FU) in wild-type (WT), PKO , interleukin (IL)-10-/- and properdin/IL-10-/- double knock-out (DKO) mice. At the time of euthanasia their jejunum was collected for histology, immunohistochemistry and cytokine and complement activation measurements. Complement became activated in mice receiving 5-FU, indicated by increased intestinal levels of C3a and C5a. Compared to WT, PKO mice experienced significantly less mucositis, despite C3a levels as high as inflamed WT mice and slightly less C5a. Conversely, PKO mice had higher intestinal levels of IL-10. IL-10 expression was mainly by epithelial cells in both uninflamed and inflamed PKO mice. IL-10-/- mice proved to be highly susceptible to mucositis and DKO mice were equally susceptible, demonstrating that a lack of properdin does not protect mice lacking IL-10. We interpret our findings to indicate that, to a significant extent, the inflammation of mucositis is properdin-dependent but complement activation-independent. Additionally, the benefit achieved in the absence of properdin is associated with increased IL-10 levels, and IL-10 is important in limiting mucositis.

Altered expression of metabolic proteins and adipokines in patients with amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is an adult-onset neurodegenerative disease characterized by the loss of upper cortical and lower motor neurons. ALS causes death within 2-5years of diagnosis. Diet and body mass index influence the clinical course of disease, however there is limited information about the expression of metabolic proteins and fat-derived cytokines (adipokines) in ALS. In healthy controls and subjects with ALS, we have measured levels of proteins and adipokines that influence metabolism. We find altered levels of active ghrelin, gastric inhibitory peptide (GIP), pancreatic polypeptide (PP), lipocalin-2, plasminogen activator inhibitor-1 (PAI-1), interleukin-6 (IL-6) and 8 (IL-8), and tumor necrosis factor alpha (TNFα) in the plasma of ALS patients relative to controls. We also observe a positive correlation between the expression of plasma nerve growth factor (NGF) relative to disease duration, and an inverse correlation between plasma glucagon and the ALS functional rating scale-revised (ALSFRS-R). Further studies are required to determine whether altered expression of metabolic proteins and adipokines contribute to motor neuron vulnerability and how these factors act to modify the course of disease.

Elevation of the terminal complement activation products C5a and C5b-9 in ALS patient blood.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease, characterized by the progressive loss of motor neurons within the central nervous system. Neural degeneration and inflammatory processes, including activation of the complement system are hallmarks of this pathology. Our past work in ALS animal models (hSOD1 G93A rodents) has revealed that blockade of the receptor for complement activation fragment C5a (C5aR), improves ALS-like symptoms and extends survival. We now show that the levels of C5a and C5b-9, but not C3a nor C4a, are significantly elevated in plasma from ALS patients compared to healthy controls. C5a was also elevated within leukocytes from ALS patients suggesting heightened C5a receptor interaction. Overall, these findings indicate that there is enhanced peripheral immune complement terminal pathway activation in ALS, which may have relevance in the disease process.

The C5a receptor antagonist PMX205 ameliorates experimentally induced colitis associated with increased IL-4 and IL-10.

BACKGROUND AND PURPOSE: Anti-complement therapies have not been advanced for treating the inflammatory bowel diseases (IBDs) despite a growing body of evidence that blocking C5a protects against induced colitis in rodents. The purpose of this study was to further build on this evidence by examining the efficacy, mechanism and specificity of a potent, non-competitive and orally active C5a receptor (CD88) antagonist, PMX205, in the dextran sulphate sodium (DSS) model of murine innate colitis. EXPERIMENTAL APPROACH: Mice with DSS added to their drinking water were orally administered 100 or 200 µg day(-1) PMX205 in prophylactic and therapeutic regimens. Clinical illness, colon histology and local generation of inflammatory mediators were measured to evaluate the impact of PMX205 on disease. KEY RESULTS: PMX205 significantly prevented DSS-induced colon inflammation in both regimens, associated with lower pro-inflammatory cytokine production and nitrotyrosine staining in colon sections. Additionally, the levels of anti-inflammatory cytokines IL-4 and IL-10 were increased. PMX205 had no significant effect on C5a levels. The beneficial effect of PMX205 was seen in two strains of mice of differing sensitivities to DSS inflammation, but was inactive in mice lacking CD88. CONCLUSIONS AND IMPLICATIONS: Pharmacological inhibition of C5a activity by PMX205 is efficacious in preventing DSS-induced colitis, providing further evidence that targeting CD88 in IBD patients could be a valuable therapeutic option.

Oral treatment with complement factor C5a receptor (CD88) antagonists inhibits experimental periodontitis in rats.

BACKGROUND AND OBJECTIVE: The complement activation product 5a (C5a) is a potent mediator of the innate immune response to infection, and may thus also importantly determine the development of periodontitis. The present study was designed to explore the effect of several novel, potent and orally active C5a receptor (CD88) antagonists (C5aRAs) on the development of ligature-induced periodontitis in an animal model. MATERIAL AND METHODS: Three different cyclic peptide C5aRAs, termed PMX205, PMX218 and PMX273, were investigated. Four groups of Wistar rats (n = 10 in each group) were used. Starting 3 d before induction of experimental periodontitis, rats either received one of the C5aRas (1-2 mg/kg) in the drinking water or received drinking water only. Periodontitis was assessed when the ligatures had been in place for 14 d. RESULTS: Compared with control rats, PMX205- and PMX218-treated rats had significantly reduced periodontal bone loss. CONCLUSION: The findings suggest that complement activation, and particularly C5a generation, may play a significant role in the development and progression of periodontitis. Blockade of the major C5a receptor, CD88, with specific inhibitors such as PMX205, may offer novel treatment options for periodontitis.

Neuroprotection in stroke by complement inhibition and immunoglobulin therapy.

Activation of the complement system occurs in a variety of neuroinflammatory diseases and neurodegenerative processes of the CNS. Studies in the last decade have demonstrated that essentially all of the activation components and receptors of the complement system are produced by astrocytes, microglia, and neurons. There is also rapidly growing evidence to indicate an active role of the complement system in cerebral ischemic injury. In addition to direct cell damage, regional cerebral ischemia and reperfusion (I/R) induces an inflammatory response involving complement activation and generation of active fragments, such as C3a and C5a anaphylatoxins, C3b, C4b, and iC3b. The use of specific inhibitors to block complement activation or their mediators such as C5a, can reduce local tissue injury after I/R. Consistent with therapeutic approaches that have been successful in models of autoimmune disorders, many of the same complement inhibition strategies are proving effective in animal models of cerebral I/R injury. One new form of therapy, which is less specific in its targeting of complement than monodrug administration, is the use of immunoglobulins. Intravenous immunoglobulin (IVIG) has the potential to inhibit multiple components of inflammation, including complement fragments, pro-inflammatory cytokine production and leukocyte cell adhesion. Thus, IVIG may directly protect neurons, reduce activation of intrinsic inflammatory cells (microglia) and inhibit transendothelial infiltration of leukocytes into the brain parenchyma following an ischemic stroke. The striking neuroprotective actions of IVIG in animal models of ischemic stroke suggest a potential therapeutic potential that merits consideration for clinical trials in stroke patients.

Role of complement C5a in mechanical inflammatory hypernociception: potential use of C5a receptor antagonists to control inflammatory pain.

BACKGROUND AND PURPOSE: C5a, a complement activation product, exhibits a broad spectrum of inflammatory activities particularly neutrophil chemoattraction. Herein, the role of C5a in the genesis of inflammatory hypernociception was investigated in rats and mice using the specific C5a receptor antagonist PMX53 (AcF-[OP(D-Cha)WR]). EXPERIMENTAL APPROACH: Mechanical hypernociception was evaluated with a modification of the Randall-Selitto test in rats and electronic pressure meter paw test in mice. Cytokines were measured by ELISA and neutrophil migration was determined by myeloperoxidase activity. KEY RESULTS: Local pretreatment of rats with PMX53 (60-180 microg per paw) inhibited zymosan-, carrageenan-, lipopolysaccharide (LPS)- and antigen-induced hypernociception. These effects were associated with C5a receptor blockade since PMX53 also inhibited the hypernociception induced by zymosan-activated serum and C5a but not by the direct-acting hypernociceptive mediators, prostaglandin E(2) and dopamine. Underlying the C5a hypernociceptive mechanisms, PMX53 did not alter the cytokine release induced by inflammatory stimuli. However, PMX53 inhibited cytokine-induced hypernociception. PMX53 also inhibited the recruitment of neutrophils induced by zymosan but not by carrageenan or LPS, indicating an involvement of neutrophils in the hypernociceptive effect of C5a. Furthermore, the C5a-induced hypernociception was reduced in neutrophil-depleted rats. Extending these findings in rats, blocking C5a receptors also reduced zymosan-induced joint hypernociception in mice. CONCLUSIONS AND IMPLICATIONS: These results suggest that C5a is an important inflammatory hypernociceptive mediator, acting by a mechanism independent of hypernociceptive cytokine release, but dependent on the presence of neutrophils. Therefore, we suggest that inhibiting the action of C5a has therapeutic potential in the control of inflammatory pain.

Species dependence for binding of small molecule agonist and antagonists to the C5a receptor on polymorphonuclear leukocytes.

This study investigated the receptor binding affinities of a C5a agonist and cyclic antagonists for polymorphonuclear leukocytes (PMNs) isolated from human, sheep, pig, dog, rabbit, guinea pig, rat and mouse. The affinities of the two small molecule antagonists, F-[OPdChaWR] and AcF-[OPdChaWR], and the agonist, YSFKPMPLaR, revealed large differences in C5a receptor (C5aR) affinities between species. The antagonists bound to human, rat and dog PMNs with similar high affinities, but with lower affinities to PMNs from all other species. The C5a agonist also bound with varying affinities between species, but showed a different affinity profile to the antagonists. In contrast, recombinant human C5a had similar affinity for PMNs of all species investigated. The low correlation between the affinities of the antagonists and the agonist between species either suggests that different receptor residues are important for distinguishing between agonist/antagonist binding, or that the agonist and antagonist peptides bind to two distinct sites within the C5aR.

Modulation of ligand selectivity by mutation of the first extracellular loop of the human C5a receptor.

The cyclic C5a receptor antagonist, phenylalanine [L-ornithine-proline-D-cyclohexylalanine-tryptophan-arginine] (F-[OPchaWR]), has approximately 1000-fold less affinity for the C5a receptor (C5aR) on murine polymorphonuclear leukocytes than on human. Analysis of C5aR from different species shows that a possible cause of this difference is the variation in the sequence of the first extracellular loop of the receptor. The mouse receptor contains Y at a position analogous to P(103) in the human receptor, and D at G(105). To test this hypothesis, we expressed human C5aR mutants (P(103)Y, G(105)D and the double mutant, P(103)Y/G(105)D) in RBL-2H3 cells and investigated the effects of these mutations on binding affinity and receptor activation. All three mutant receptors had a higher affinity for human C5a than the wild-type receptor, but showed no significant difference in the ability of F-[OPchaWR] to inhibit human C5a binding. However, all of the mutant receptors had substantially lower affinities for the weak agonist, C5a des Arg(74) (C5adR(74)), and two altered receptors (G(105)D and P(103)Y/G(105)D) had much lower affinities for the C-terminal C5a agonist peptide analogue, L-tyrosine-serine-phenylalanine-lysine-proline-methionine-proline-leucine-D-alanine-arginine (YSFKPMPLaR). Although it is unlikely that differences at these residues are responsible for variations in the potency of F-[OPchaWR] across species, residues in the first extracellular loop are clearly involved in the recognition of both C5a and C5a agonists. The complex effects of mutating these residues on the affinity and response to C5a, C5adR(74), and the peptide analogues provide evidence of different binding modes for these ligands on the C5aR.

A new small molecule C5a receptor antagonist inhibits the reverse-passive Arthus reaction and endotoxic shock in rats.

C5a is implicated as a pathogenic factor in a wide range of immunoinflammatory diseases, including sepsis and immune complex disease. Agents that antagonize the effects of C5a could be useful in these diseases. We have developed some novel C5a antagonists and have determined the acute anti-inflammatory properties of a new small molecule C5a receptor antagonist against C5a- and LPS-induced neutrophil adhesion and cytokine expression, as well as against some hallmarks of the reverse Arthus reaction in rats. We found that a single i.v. dose (1 mg/kg) of this antagonist inhibited both C5a- and LPS-induced neutropenia and elevated levels of circulating TNF-alpha, as well as polymorphonuclear leukocyte migration, increased TNF-alpha levels and vascular leakage at the site of immune complex deposition. These results indicate potent anti-inflammatory activities of a new C5a receptor antagonist and provide more evidence for a key early role for C5a in sepsis and the reverse Arthus reaction. The results support a role for antagonists of C5a receptors in the therapeutic intervention of immunoinflammatory disease states such as sepsis and immune complex disease.