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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online October 16, 2003 as doi:10.1096/fj.02-1103fje. |
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* Brain Inflammation and Immunity Group (BIIG) and Complement Biology Group, Department of Medical Biochemistry and Immunology, University of Wales College of Medicine (UWCM), Cardiff, UK;
Department of Medicine, Section of Endocrinology, Diabetes, and Metabolism, UWCM, Cardiff, UK;
Choju Medical Institute, Fukushimura Hospital, Toyohashi, Japan; and
Section of Neurology, Division of Genomic Medicine, University of Sheffield Medical School, Sheffield, UK
2Correspondence: Brain Inflammation and Immunity Group, Department of Medical Biochemistry, University of Wales College of Medicine, Tenovus Bldg., Heath Park, Cardiff, CF14 4XN, UK. E-mail: Gasque{at}cardiff.ac.uk
SPECIFIC AIM
Traditionally the unique high-affinity, G-protein-coupled seven transmembrane receptor for C3a anaphylatoxin (C3aR) was thought to be present only on myeloid cells, but the identification of abundant C3aR message in the adrenal gland, pituitary, and central nervous system suggests a much greater role in the pathogenesis of inflammatory and tissue homeostasis than previously thought.
These novel observations led us to investigate the cellular and molecular pathways that could link C3aR expression in endocrine tissues with newly discovered anti-inflammatory activities of the innate immune complement C3a cytokine.
PRINCIPAL FINDINGS
1. C3aR is expressed in several key endocrine tissues
Two-way communication exists between the endocrine and immune systems in phylogenetically diverse animals using molecules like hormones and cytokines. Here we describe a new pathway by which the innate immune protein C3a, a complement-derived cytokine, stimulates anterior pituitary hormone release and activates the hypothalamic-pituitary-adrenal axis, a reflex central to the stress response and to the control of inflammation.
Analysis of RT-PCR and Southern blot experiments revealed expression of C3aR mRNA in various rat tissues including spinal cord, lung, heart, liver, spleen, kidney, and muscle, with particularly high levels in endocrine tissues (i.e., adrenal, ovary, and testis). These results were confirmed by Northern blot (data not shown). Routine immunoperoxidase cytochemistry for C3aR using the affinity-purified antibody in wax-embedded rat sections demonstrated the presence of C3aR protein in several endocrine tissues together with lung, skin, spleen, kidney, and heart, but not liver (data not shown). A discrete specific staining was also localized to a subset of neural cells in the hypothalamus. We used the anti-rat C3aR antibody with different anti-hormone specific antibodies to perform double immunofluorescence stainings of rat pituitary frozen sections. These showed consistent and specific staining for C3aR in the anterior lobe, robust staining in the intermediate lobe, and virtually no detectable signal in the posterior lobe. C3aR was expressed in subpopulations of somatotrophs (GH) and thyrotrophs (TSH) and in the majority of lactotrophs (PRL) and corticotrophs (ACTH) (Fig. 1
). Positive staining was abolished when the primary antibody was omitted or preadsorbed with C3aR loop peptide. Why only some cells of each type express C3aRs is unclear; similar patterns of expression were observed in pituitary cell lines GH3 and AtT20 (data not shown). We also found complete colocalization of C3aR and S-100ß protein, a marker of nonendocrine agranular folliculostellate cells. These enigmatic parenchymal dendritic-like cells are thought to be key functional integration units of a dynamically active cell network wiring the whole pituitary gland and are known to express several pattern recognition molecules (e.g., TLR4 and CD14).
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2. Physiological doses of recombinant C3a control pituitary hormone secretion in vitro
Given the strong C3aR staining in rat pituitary, we examined whether physiological concentrations of recombinant human C3a (free of bacterial lipopolysaccharide) could control the release of immunoregulatory pituitary hormones. Enzymatically dispersed rat anterior pituitary tissues were incubated with a single dose (100 nM) of C3a and hormone levels were recorded between 5 min and 6 h. ACTH, PRL, GH, and TSH secretion in the same experiments were measured by radioimmunoassay. Basal secretion of ACTH, PRL, and GH but not TSH increased slightly with time, reaching significance at 6 h. The response to C3a stimulation was particularly strong and rapid as PRL, GH, and ACTH levels rose significantly above basal levels within 5 min of exposure. The degrees of stimulation by C3a compared with basal values were, at 4 h: 1- to 2-fold for ACTH (P<0.01), 3- to 6-fold for PRL (P<0.001), and a remarkable 9- to 15-fold increase for GH (P<0.001). Four additional experiments confirmed the specific and selective nature of C3a activities. Surprisingly, we could detect no alterations in TSH secretion.
When different doses of C3a were applied to pituitary cultures for 4 h, there was a dose-related stimulation of GH, PRL, and ACTH secretion; at 250 nM the increases over basal levels were 6- to 7-fold, 3- to 4-fold, and 1.5-fold respectively (Fig. 2
). Remarkably, the stimulation of hormone secretion was consistently blocked by pretreating the cells with pertussis toxin (PT, 150 ng/mL), suggesting the role of a C3a receptor coupled to G
i-like G-proteins (Fig. 2)
. These functional in vitro studies provide unique and strong evidence that C3a, produced notably during sustained systemic inflammation, is a potent stimulus for GH, PRL, and ACTH release.
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3. A functionally inactive form of C3a (C3adesArg) is a potent activator of pituitary hormone secretion independent of the Gi protein-coupled C3a receptor pathway
C3adesArg (also known as the acylation-stimulating protein) is almost the exclusive form of C3a detectable in the human circulation following the rapid and highly efficient inactivation by serum carboxypeptidase N. It is well documented that cleavage of the carboxy terminal arginine results in a lack of 1) binding and signaling of C3adesArg to the GI protein-coupled, high-affinity C3aR and 2) activation of phagocytes, smooth muscle cells, and mast cells.
However, we here report for the first time that C3adesArg is at least equipotent at stimulating hormone release and that PT is unable to inhibit this response (Fig. 2)
. These original findings will certainly provide a unique biochemical framework to isolate and characterize the molecular signature of the enigmatic C3adesArg receptor. A novel C3adesArg receptor (C5L2) has recently been identified on adipocytes and is involved in the control of lipid metabolism.
4. Physiological doses of C3a control hormone secretion in vivo
To further explore the cellular and molecular routes involved in C3a activities to control hormone release, particularly in organs of the HPA stress axis, we performed intravenous injections of either recombinant C3a (single dose, 400 ng/mL of blood, ie. 9–10 µg/rat, four animals per group; n=5), recombinant C3adesArg (dose as above), or control saline solution into male rats and measured levels of GH, PRL ACTH, and corticosterone over 60 min. C3a had a pronounced effect on the circulating levels of all hormones tested; peak increases (expressed as % of basal at time 0 for treated vs. saline controls) were for GH (30 min: 172±42%, P<0.05), PRL (45 min: 291±69%, P<0.05), ACTH (45 min: 233±8%, P<0.01), and corticosterone (60 min: 204±22%, P<0.01). Circulating levels of PRL, GH, and corticosterone were also rapidly up-regulated after C3adesArg injection.
Although the significance of elevated levels of GH and PRL in inflammation is not understood entirely, the current paradigm is that both PRL and GH (and IGF-1 and thyroid hormones) acting on lymphoid tissues and circulating immune cells play important immunoregulatory roles. Moreover, it is clear that increased levels of ACTH will promote the release of glucocorticoids from the adrenal gland, which in turn are known to have robust immunoregulatory activities. Hence, we propose that high levels of complement-derived C3a and/or C3adesArg cytokines produced when the organism is subjected to major immune inflammatory reactions (e.g., sepsis) engage a critical feedback mechanism with the release of immunoregulatory hormones to safely return the immune system to homeostasis. This paradigm is supported by pioneering studies of C3aR knockout mice which indicated a protective role of C3a in a model of sepsis.
CONCLUSIONS AND SIGNIFICANCE
Although the complement-mediated proinflammatory responses are crucial to our body’s well-being, a too strong response can be harmful and lead to devastating illnesses (e.g., arthritis, sepsis). We describe here a new pathway by which complement C3a and its immune inactive metabolite C3adesArg stimulate pituitary hormone release and activate the hypothalamic-pituitary-adrenal axis (HPA), a reflex central to the stress response and to the negative control of inflammation. We show that C3a receptors are expressed in the pituitary and that C3a and C3adesArg stimulate release of prolactin, growth hormone, and adrenocorticotrophin, in vitro and in vivo. PT blocks the response to C3a but not C3adesArg, indicating the plausible presence of two receptors. Identification and molecular characterization of the novel C3adesArg receptor are now highly warranted.
In conclusion, we propose that molecules of the complement system, in addition to promoting inflammation (leukocyte activation, phagocytosis, degranulation of mast cells), are capable of modulating systemic responses through communication with the endocrine pituitary/adrenal glands. From an evolutionary point of view, we may have unveiled an ancient cross-talk between the innate immune and endocrine systems to control safe tissue homeostasis.
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FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-1103fje; doi: 10.1096/fj.02-1103fje 
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