| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
|
FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online March 9, 2005 as doi:10.1096/fj.04-2797fje. |
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
* Department of Pathology, School of Dental Medicine,
Pulmonary, Allergy and Critical Care Division, Department of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA
1 Correspondence: Department of Pathology, University of Pennsylvania, School of Dental Medicine, 240 S. 40th St., Philadelphia, PA, 19104-6002, USA. E-mail: ali{at}path.dental.upenn.edu
SPECIFIC AIMS
Studies with animal models and immunohistochemistry analysis of lung tissue indicate that anaphylatoxins (C3a and C5a) may regulate airway hyperresponsiveness (AHR) in asthma via activation of their cell surface G-protein-coupled receptors in airway smooth muscle (ASM) cells. The purpose of this study was to test the hypothesis that C3a and C5a receptors are expressed in cultured primary human and murine ASM cells and to determine the signal transduction pathways whereby the functions of these receptors are regulated.
PRINCIPAL FINDINGS
1. Mast cells and monocytes/macrophages express C3a and C5a receptors (C3aR and C5aR) but ASM cells do not
C3aR and C5aR are endogenously expressed in human mast cells and circulating leukocytes. RT-PCR was performed to determine whether mRNA for these receptors is present in cultured human ASM cells. A human mast cell line, HMC-1, purified human monocytes that constitutively express C3aR and C5aR, served as a control. As shown in Fig. 1
A, C3aR and C5aR mRNA was expressed by HMC-1 cells and human monocytes. mRNA for these receptors was undetectable in human ASM cells. Flow cytometric analysis using C3aR-specific antibody demonstrated the presence of C3aR on the surface of mast cells but not ASM cells.
|
C3a and C5a mediate their biological response via activation of G-protein-coupled receptors, which activate phospholipase Cß and mobilize intracellular Ca2+. Using Ca2+ mobilization, we found that C3a and C5a caused Ca2+ mobilization in HMC-1 cells. Anaphylatoxins, however, did not stimulate Ca2+ responses in human ASM cells. For control, we tested the effect of bradykinin, which activates Gq protein-coupled receptors on human ASM cells, to stimulate Ca2+ mobilization. ASM cells that were unresponsive to C3a or C5a for Ca2+ mobilization responded to bradykinin robustly (data not shown). These data demonstrate that human mast cells but not ASM cells express functional C3aR and C5aR.
Studies in animal models indicated that the effects of C3a in asthma are mediated via the activation of its receptors in ASM cells. We used RT-PCR to assess the expression of C3aR and C5aR mRNA in primary cultures of murine ASM cells. Bone marrow-derived macrophages and an alveolar macrophage cell line MH-S were used for comparison. As shown in Fig. 1B
, macrophages expressed C3aR and C5aR mRNA, but mRNA for these receptors could not be detected in murine ASM cells. We found that C3a and C5a mobilized cytosolic Ca2+ in bone marrow-derived macrophages, but C3a did not stimulate Ca2+ mobilization in murine ASM cells even though bradykinin stimulated a robust Ca2+ response (data not shown).
To examine whether a correlation exists between C3aR expression in vivo and in vitro, immunohistochemical analysis of C3aR expression in human trachea and bronchus was performed. The data demonstrate that C3aR is not expressed in normal smooth-muscle positive cells of human trachea and bronchus and suggest that the lack of C3aR in cultured human airway smooth muscle is not associated with loss of C3aR expression due to cell culture.
2. Proinflammatory mediators do not induce C3aR expression in human ASM cells
ASM cells respond to proinflammatory mediators (lipopolysaccharide, LPS) and cytokines such as interleukin-1ß (IL-1ß) for transcription factor activation and cytokine production, raising the possibility that C3aR could be expressed in ASM cells de novo upon stimulation with inflammatory mediators and cytokines. To test this, we cultured ASM cells with LPS (100 ng/mL) or IL-1ß (10 ng/mL) for 24 h and assayed for the production of proinflammatory cytokines in culture supernatants and C3aR expression on the cell surface. We found that unstimulated ASM cells produced 32.2 ± 5 pg/mL of IL-8 in culture supernatant. LPS and IL-1ß induced 253.5 ± 10 pg/mL and 156 ± 3 pg/mL of IL-8 production, respectively. However, we could not detect the expression of C3aR on the surface of ASM cells by immunostaining. C3a failed to induce intracellular Ca2+ mobilization in LPS or IL-1ß-treated ASM cells. These findings provide additional support for the contention that human ASM cells do not endogenously express C3aR and that proinflammatory mediators do not induce their expression.
3. Human ASM enhance C3a-induced mast cell degranulation following cell-cell contact
Asthmatic patients have increased numbers of mast cells in the smooth muscle layer of airways vs. normal subjects. Degranulated mast cells are detected in greater number in ASM of patients who died from asthma than in nonasthmatic controls. We found that a low concentration of C3a (10 nM) caused robust mast cell degranulation (Fig. 2
A). Coculture of mast cells with ASM cells for 24 h resulted in significant enhancement of C3a-induced response. To test the possibility that factors released from ASM cells enhance C3a-induced mast cell degranulation, we incubated mast cells for 24 h with ASM culture supernatant. In contrast to ASM cells, its culture supernatant had no effect on C3a-induced mast cell degranulation. Coculture of ASM cells and mast cells for 15 min was sufficient to enhance C3a-induced mast cell degranulation (Fig. 2B
). These findings suggest that rapid cell-cell contact between mast cells and ASM cells is required for enhanced C3a-induced mast cell mediator release. In addition, molecules on ASM cells that cross-regulate mast cell function are constitutively expressed on their cell surface.
|
4. Constitutively expressed stem cell factor on ASM cells is not required for enhancement of C3a-induced mast cell degranulation
Stem cell factor (SCF) is a potent mast cell chemoattractant that regulates their growth, function, and survival. Using flow cytometry, we demonstrated that SCF is present on the surface of ASM cells but not mast cells. To explore the possibility that ASM cell-derived SCF could interact with its receptors c-kit on mast cells to enhance C3a-induced degranulation, we preincubated ASM cells with a neutralizing antibody to SCF (10 µg/mL, 1 h) and mast cells with a neutralizing antibody to c-kit (10 µg/mL, 1 h). We found that these antibodies had no effect on the enhancement of C3a-induced mast cell degranulation by ASM cells. These findings suggest that cross-regulation of C3a-induced mast cell degranulation by ASM cells involves molecules other than SCF.
5. Dexamethasone-treated ASM cells are less effective than untreated cells in enhancing C3a-induced mast cell degranulation
We treated ASM cells with dexamethasone (10 and 100 nM for 16 h) and tested its effects on mast cell function. We found that treatment of ASM cells with dexamethasone had no effect on cell surface expression of SCF. However, steroid-treated ASM cells were significantly less effective than untreated cells in enhancing C3a-induced mast cell degranulation.
CONCLUSIONS AND SIGNIFICANCE
This study demonstrates the surprising observation that whereas mast cells express functional C3aR and C5aR, human and murine ASM cells do not. It reveals the novel finding that cell-cell interaction between mast cells and ASM cells enhances C3a-induced mast cell degranulation, providing a possible mechanism for the roles of anaphylatoxins in the pathogenesis of allergic asthma.
Earlier attempts to determine whether or not ASM cells express functional receptors for anaphylatoxins yielded contradictory findings. This study clearly demonstrates that cultured human and murine ASM cells do not express C3aR and C5aR. Cytokines that activate ASM cells to cause the release of proinflammatory mediators do not induce C3aR in ASM cells. Most importantly, we could not detect C3aR in smooth muscle-positive cells of human trachea or bronchus. Based on the studies described herein, we propose that ASM cells do not express anaphylatoxin receptors in vivo but the presence of these receptors in ASM tissue reported by some investigators reflects the recruitment of mast cells or other inflammatory cells into the airway. This view is supported by the following observations. 1) C3a and C5a do not cause contraction of isolated murine tracheal strips and fail to induce AHR or airway inflammation after intratracheal instillation in naive mice. In contrast, in mice immunized with house dust mite subsequent intratracheal administration of C3a and C5a induce AHR and airway inflammation. 2) Mast cell-deficient mice do not develop AHR after exposure to aerosolized allergen in the absence of adjuvant. When bone marrow-derived mast cells are transferred to mast cell-deficient mice, they migrate to the tracheal tissue of the recipient mice to restore AHR after allergen exposure. 3) Studies with human subjects have provided evidence for a close interaction between mast cells and ASM cells in asthma. Thus, immunohistological analysis of biopsy specimens from subjects with asthma revealed a striking increase in the number of mast cells among airway smooth muscle bundles vs. those from normal subjects.
The novel and most interesting finding of the present study was that coculture of mast cells with ASM cells resulted in enhancement of C3a-induced mast cell degranulation. The demonstration that incubation of mast cells with ASM cell culture supernatants failed to enhance mast cell degranulation suggests that cell-cell contact between ASM cells and mast cells are likely required for enhanced mast cell degranulation. The demonstration that neutralizing antibodies to SCF and c-kit had no effect on the enhancement of C3a-induced mast cell degranulation indicates that another receptor-ligand association is responsible for this interaction. This view is supported by the finding that dexamethasone-treated ASM cells were normal for cell surface SCF expression but significantly less effective in enhancing C3a-induced mast cell degranulation compared with untreated cells. Human ASM cells and mast cells express a variety of adhesion molecules and their counter receptors. Mast cells express a novel adhesion molecule, spermatogenic immunoglobulin superfamily (SgIgSF). Whether these or other molecules on ASM cells and mast cells interact to enhance C3a-induced mast cell degranulation remains to be determined.
We made the novel observation that ASM cells enhance C3a-induced mast cell degranulation after cell-cell contact, which likely regulates ASM function, contributing to the pathogenesis of asthma (Fig. 3
). Glucocorticoid dexamethasone used to treat asthma substantially inhibited the ability of ASM cells to enhance mast cell degranulation.
|
FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-2797fje;
Related Articles
FASEB J 2006 20: 199.
FASEB J 2005 19: 1585.
This article has been cited by other articles:
|
|
 |
|
  R. A. Panettieri Jr., M. I. Kotlikoff, W. T. Gerthoffer, M. B. Hershenson, P. G. Woodruff, I. P. Hall, and S. Banks-Schlegel Airway Smooth Muscle in Bronchial Tone, Inflammation, and Remodeling: Basic Knowledge to Clinical Relevance Am. J. Respir. Crit. Care Med., February 1, 2008; 177(3): 248 - 252. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. K. Zaidi, Y. Amrani, R. A. Panettieri, and H. Ali Response to C3a, mast cells, and asthma FASEB J, February 1, 2006; 20(2): 199 - 199. [Full Text] [PDF]
|
|
|
|
|
|
P. Bradding C3a, mast cells, and asthma FASEB J, October 1, 2005; 19(12): 1585 - 1585. [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
