Edimara S. Reis

The Role of the Anaphylatoxins in Health and Disease

The anaphylatoxin (AT) C3a, C5a and C5a-desArg are generally considered pro-inflammatory polypeptides generated after proteolytic cleavage of C3 and C5 in response to complement activation. Their well-appreciated effector functions include chemotaxis and activation of granulocytes, mast cells and macrophages. Recent evidence suggests that ATs are also generated locally within tissues by pathogen-, cell-, or contact system-derived proteases. This local generation of ATs is important for their pleiotropic biologic effects beyond inflammation. The ATs exert most of the biologic activities through ligation of three cognate receptors, i.e. the C3a receptor, the C5a receptor and the C5a receptor-like, C5L2. Here, we will discuss recent findings suggesting that ATs regulate cell apoptosis, lipid metabolism as well as innate and adaptive immune responses through their impact on antigen-presenting cells and T cells. As we will outline, such regulatory functions of ATs and their receptors play important roles in the pathogenesis of allergy, autoimmunity, neurodegenerative diseases, cancer and infections with intracellular pathogens.

Clinical Aspects and Molecular Basis of Primary Deficiencies of Complement Component C3 and its Regulatory Proteins Factor I and Factor H

The complement system participates in both innate and acquired immune responses. Deficiencies in any of the protein components of this system are generally uncommon and require specialized services for diagnosis. Consequently, complement deficiencies are clinically underscored and may be more common than is normally estimated. As C3 is the major complement component and participates in all three pathways of activation, it is fundamental to understand all the clinical consequences observed in patients for which this protein is below normal concentration or absent in the serum. C3 deficiencies are generally associated with higher susceptibility to severe infections and in some cases with autoimmune diseases such as systemic lupus erythematosus. Here, we review the main clinical aspects and the molecular basis of primary C3 deficiency as well as the mutations in the regulatory proteins factor I and factor H that result in secondary C3 deficiencies. We also discuss the use of animal models to study these deficiencies.

C5a receptor-deficient dendritic cells promote induction of Treg and Th17 cells

C5a is a proinflammatory mediator that has recently been shown to regulate adaptive immune responses. Here we demonstrate that C5a receptor (C5aR) signaling in DC affects the development of Treg and Th17 cells. Genetic ablation or pharmacological targeting of the C5aR in spleen‐derived DC results in increased production of TGF‐β leading to de novo differentiation of Foxp3+ Treg within 12 h after co‐incubation with CD4+ T cells from DO11.10/RAG2−/− mice. Stimulation of C5aR−/− DC with OVA and TLR2 ligand Pam3CSK4 increased TGF‐β production and induced high levels of IL‐6 and IL‐23 but only minor amounts of IL‐12 leading to differentiation of Th cells producing IL‐17A and IL‐21. Th17 differentiation was also found in vivo after adoptive transfer of CD4+ Th cell into C5aR−/− mice immunized with OVA and Pam3CSK4. The altered cytokine production of C5aR−/− DC was associated with low steady state MHC class II expression and an impaired ability to upregulate CD86 and CD40 in response to TLR2. Our data suggest critical roles for C5aR in Treg and Th17‐cell differentiation through regulation of DC function.

Sleep and circadian rhythm regulate circulating complement factors and immunoregulatory properties of C5a

The sleep-wake cycle is characterized by complex interactions among the central nervous, the endocrine and the immune systems. Continuous 24-h wakefulness prevents sleep-associated hormone regulation resulting in impaired pro-inflammatory cytokine production. Importantly, cytokines and hormones also modulate the complement system, which in turn regulates several adaptive immune responses. However, it is unknown whether sleep affects the activation and the immunoregulatory properties of the complement system. Here, we determined whether the 24-h sleep-wake cycle has an impact on: (i) the levels of circulating complement factors; and (ii) TLR4-mediated IL-12 production from human IFN-γ primed monocytes in the presence or absence of C5a receptor signaling. For this purpose, we analyzed the blood and blood-derived monocytes of 13 healthy donors during a regular sleep-wake cycle in comparison to 24 h of continuous wakefulness. We found decreased plasma levels of C3 and C4 during nighttime hours that were not affected by sleep. In contrast, sleep was associated with increased complement activation as reflected by elevated C3a plasma levels during nighttime sleep. Sleep deprivation prevented such activation. At the cellular level, C5a negatively regulated TLR4-mediated IL-12p40 and p70 production from human monocytes. Importantly, this regulatory effect of C5a on IL-12p70 production was effective only during daytime hours. Thus, similar to hormones, some complement factors and immunoregulatory properties of C5a are influenced by sleep and the circadian rhythm. Our findings that continuous wakefulness has a negative impact on complement activation may provide a rationale for the immunosupportive functions of sleep.

Human monocyte-derived dendritic cells are a source of several complement proteins

Abstract.ObjectiveLittle is known about the role of local production of complement components by dendritic cells (DCs) during the generation of specifi c immune responses. In this study, we demonstrate that human DCs are an extrahepatic source of several soluble complement proteins.MethodsReverse transcriptase polymerase chain reaction (RT-PCR) and Western blot were used to evaluate the expression and production of several complement proteins.ResultsWe show that DCs produce C3, C5, C9, Factor (F)I, FH, FB, FD and properdin at levels similar to macrophages. Treatment of DCs with lipopolysaccharide (LPS) promoted an increase in the expression of C3 and FI mRNAs and a decrease in C5 mRNA, while C9, FH, FB, FD and properdin mRNA levels were not affected. Treatment with interleukin (IL) −1 or dexamethasone induced a modest increase in C3 mRNA levels and did not affect the expression of other complement components.ConclusionDCs are a source of complement proteins whose synthesis may be regulated in response to different infl ammatory stimuli.

Homozygous hereditary C3 deficiency due to a premature stop codon.

C3 deficiency in humans is a rare disorder characterized by severe recurrent infections. We identified the mutations responsible for a complete homozygous C3 deficiency. Sequencing of the proband C3 cDNA (5067 bp) revealed the following alterations: (a) a silent G→A transition at nucleotide 972; (b) a T→C substitution at nucleotide 1001 resulting in a L314P transition; and (c) a stop codon in exon 13 caused by a G→A substitution at position 1716. The presence of the same premature termination codon was confirmed in approximately half the clones obtained from the proband’s paternal and maternal genomic DNAs. Finally, the proband produced ∼20-fold less C3 mRNA than the normal control. Therefore, in addition to the fact that no functional protein will be synthesized in the deficient cells, this nonsense mutation may be associated with the low C3 mRNA levels.

Hereditary Human Complement C3 Deficiency Owing to Reduced Levels of C3 mRNA

An 8‐year‐old son (L.A.S.) of consanguineous parents, presented recurrent bacterial infections, vasculitis and extremely low levels of serum C3 (0.15 µg/ml). The classical and alternative pathway haemolytic activities and the generation of opsonins and chemotactic factors derived from the activation of the complement system were markedly affected in the probands serum. An in vitro addition of purified C3 restored the classical pathway‐dependent haemolytic activity of his serum. Autoradiographs of the probands lipopolysaccharide (LPS)‐stimulated and 35S‐labelled fibroblast supernatants after that the SDS‐PAGE revealed no C3 α or β chains. The amount of C3 mRNA synthesized by the probands fibroblasts, as evaluated by reverse transcription–polymerase chain reaction (RT–PCR) assays, was greatly reduced.

Nonsense-codon-mediated decay in human hereditary complement C3 deficiency

C3 occupies a central position in the complement pathway, mediating such diverse functions as convertase activity, opsonization and anaphylotoxin production. The deficiency of this protein is a rare autosomal recessive inherited disease, characterized by severe recurrent infections and immune complex disorders. We looked for molecular alterations that could explain the C3 deficiency present in a Brazilian boy of consanguineous parents who suffered from recurrent bacterial infections. Using reverse-transcriptase polymerase chain reaction to amplify C3 mRNA from LPS-stimulated fibroblasts from the patient, we demonstrated that his C3 gene has no large structural aberrations. However, after sequencing the amplified and cloned products we found: (1) a L314P amino acid substitution; (2) silent mutations at codons P577, S798 and A1437; and finally, (3) an R848STer substitution that results in the production of a truncated protein. Densitometry studies revealed a lower C3 mRNA concentration in the patient’s fibroblasts, suggesting an inherent instability of his C3 mRNA. Our results indicate the presence of a premature termination codon in the C3 gene that results in a lack of the protein in patient’s serum, which correlates with the acceleration of C3 mRNA decay in the patient’s fibroblasts. This mRNA instability is consistent with a nonsense-codon-mediated decay process that ensures the elimination of possible deleterious truncated proteins, which, in the case of constitutively expressed abundant proteins such as C3, may otherwise accumulate to significant levels, leading to toxicity.

Deficiency of the Human Complement Regulatory Protein Factor H Associated with Low Levels of Component C9

We identified a 4‐year‐old Brazilian boy from a family of Japanese descent and history of consanguinity, who suffered from severe recurrent pneumonia. He carries factor H (FH) deficiency associated with reduced levels of component C9 and low serum levels of C3 and factor B. His mother also presented low levels of these proteins and factor I, while his father and sister had only lower levels of FH. Western blot assays confirmed the complete absence of FH and FHL‐1 polypeptides in this patient. Sequencing of the proband’s FH cDNA revealed a homozygous G453A substitution, encoding an Arg127His change. His mother, father and sister are heterozygous for this substitution. Despite the absence of FH in the plasma, this protein was detected in the patient’s fibroblasts, suggesting that Arg127 may be important for FH secretion. Low concentrations of C9 were detected in the proband serum but no mutations in the patient’s C9 gene or promoter have been identified, suggesting that this is a consequence of uncontrolled complement activation and high C9 consumption.