Airway smooth muscle cells enhance C3a ... - The FASEB Journal

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The FASEB Journal express article10.1096/fj.04-2797fje. Published online March 9, 2005.

Airway smooth muscle cells enhance C3a-induced mast cell degranulation following cell-cell contact E. Berla Thangam,* Rampura T. Venkatesha,* Asifa K. Zaidi,* Kelly L. Jordan-Sciutto,* Dmitry A. Goncharov,† Vera P. Krymskaya,† Yassine Amrani,† Reynold A. Panettieri, Jr.,† and Hydar Ali* *Department of Pathology, School of Dental Medicine; †Pulmonary, Allergy and Critical Care Division, Department of Medicine, University of Pennsylvania, Philadelphia, PA, 19104 Corresponding author: Hydar Ali, Department of Pathology, University of Pennsylvania, School of Dental Medicine, 240 South 40th Street, Philadelphia, PA, 19104-6002. E-mail: [email protected] ABSTRACT Growing evidence suggests that anaphylatoxins, C3a and C5a, play important roles in innate immunity and may also participate in the pathogenesis of asthma. Previous studies with animal models and immunohistochemistry analysis of lung tissue indicated that anaphylatoxins may regulate airway hyperresponsiveness (AHR) in asthma via the activation of their cell surface G protein-coupled receptors (C3aR and C5aR) in airway smooth muscle (ASM) cells. Using RTPCR, flow cytometry, and confocal microscopy, we made the surprising observation that while C3aR and C5aR were expressed in human mast cells, they were not present in cultured primary human or murine ASM cells. Furthermore, we could not detect C3aR in smooth muscle-positive cells of human trachea or bronchus. Interestingly, incubation of human mast cells with ASM cells, but not its culture supernatant, caused a significant enhancement of C3a-induced mast cell degranulation. Although stem cell factor (SCF) and its receptor c-kit are constitutively expressed on ASM cells and mast cells, respectively, neutralizing antibodies to SCF and c-kit failed to inhibit ASM cell-mediated enhancement of mast cell degranulation. However, dexamethasonetreated ASM cells were normal for cell surface SCF expression but were significantly less effective in enhancing C3a-induced mast cell degranulation when compared with untreated cells. These findings suggest that cell-cell interaction between ASM cells and mast cells, via a SCF-ckit-independent but dexamethasone-sensitive mechanism, enhances C3a-induced mast cell degranulation, which likely regulates ASM function, thus contributing to the pathogenesis of asthma. Key words: complement • asthma • chemokine • cytokine

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sthma, a complex airway inflammatory disease, is characterized by bronchoconstriction, airway hyperresponsiveness (AHR) and excessive mucous production. Mast cells play a central role in the pathogenesis of asthma (1, 2). Cross-linking of high-affinity IgE

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receptors (FcεRI) on mast cells by allergen results in degranulation, leukotriene generation, and cytokine synthesis (3–7). These mediators increase vascular permeability, recruit inflammatory cells to the airway, and promote smooth muscle contraction (8–11). The complement system forms an important part of innate immunity against bacteria and other pathogens. As a system of “pattern recognition molecules”, foreign surface antigens and immune complexes initiate a proteolytic pathway leading to the formation of a lytic membrane attack complex. The anaphylatoxins C3a and C5a are generated as a byproduct of complement activation. In patients with allergic asthma, increased levels of C3a and C5a are detected in bronchoalveolar (BAL) fluid following allergic challenge (12, 13). Furthermore, C3a and C5a are potent proinflammatory mediators that interact with G protein-coupled receptors to induce mast cell chemotaxis as well as mediator release in monocytes, basophils, and eosinophils (14– 20). These findings suggest that complement components play important roles in the pathogenesis of asthma, but the mechanisms involved are not known. Recent studies in guinea pigs with a natural deficiency in C3a receptor (C3aR) expression and C3aR-deficient mice clearly demonstrated an important role of this receptor in the pathogenesis of asthma (12, 21). A surprising aspect of these studies, however, was that inflammatory airway infiltrates induced by the allergen challenge were not affected in C3aR-deleted animals. It was only the subsequent hyperresponsiveness to methacholine challenge that was reduced. Drouin et al. (22), using immunohistochemistry and in situ hybridization analysis, showed that C3aR and C5aR are expressed in airway smooth muscle (ASM) tissue of both human and mouse. Based on these findings, it was postulated that the effects of C3a on airway hyperresponsiveness may involve the direct activation of its receptors in ASM cells (12, 21). This is an attractive hypothesis given that ASM cells not only shorten in response to receptor activation but have a potential to play a more central role in asthma via the secretion of a large number of inflammatory cytokines, expression of adhesion molecules that recruit inflammatory cells, and proliferation that contributes to airway remodeling (12, 23–29). However, whether ASM cells express C3aR and C5aR and what biological functions they perform are not known. The purpose of this study was to test the hypothesis that C3aR and C5aR are expressed in ASM cells and to determine the signal transduction pathways via which these receptors activate ASM cells. For comparison, we also used human mast cells, monocytes, and murine macrophages. Surprisingly, we found that while C3aR and C5aR are expressed in leukocytes they were not present in human or murine ASM cells. We further demonstrate the novel finding that ASM cells enhance C3a-induced mast cell degranulation following cell-cell contact, which likely has important implications for understanding the mechanisms by which complement components modulate asthma and other lung diseases. MATERIALS AND METHODS Materials Purified C3a was obtained from Advanced Research Technologies (San Diego, CA). C5a, dexamethasone, and anti-smooth muscle α-actin clone 1A4 FITC conjugate antibody were purchased from Sigma (St. Louis, MO). Indo-1 AM, pluronic F-127, and Alexa Fluor 594 antirabbit IgG conjugate antibody were from Molecular Probes (Eugene, OR). PDGF was obtained


from EMD Biosciences (La Jolla, CA). Polyclonal C3aR antibody and normal rabbit IgG were obtained from Torrey Pines Biolabs (Houston, TX). Human recombinant stem cell factor was purchased from Peprotech (Rocky Hill, NJ). Anti-human SCF neutralizing antibody, anti-human c-kit neutralizing antibody, and EGF were purchased from R&D Systems (Minneapolis, MN). Goat-anti-rabbit IgG conjugated to FITC and all tissue culture reagents were purchased from Invitrogen (Gaithersburg, MD). Lipopolysaccharide (LPS, E. coli 0111.B4) was from Calbiochem (La Jolla, CA). Interleukin-1β (IL-1β) was from Roche Diagnostic (Indianapolis, IN). Cell culture and transfection For human ASM cells, tracheae were obtained from lung transplant donors in accordance with procedures approved by the University of Pennsylvania Committee on Studies Involving Human Beings. ASM cells were dissected and purified as described previously by Panettieri et al. (30). Cells were cultured in Ham’s F12 medium supplemented with 10% FBS, glutamine (2 mM), penicillin (100 U/ml), and streptomycin (100 µg/ml) and used for up to 6th passage (31). Mouse ASM cells were cultured in the same medium supplemented with PDGF (10 µg/ml) and EGF (40 µg/ml). HMC-1 cells were cultured in IMDM supplemented with 10% FCS, glutamine (2 mM), penicillin (100 U/ml), and streptomycin (100 µg/ml). A mouse alveolar macrophage cell line MH-S, obtained from ATCC, was grown in RPMI 1640 medium containing 2 mM glutamine and 10% FBS. For transfection, human ASM cells (1×106) were plated in 100 mm dishes and grown for 24 h. Cells were transfected with 40 µg of either control vector (pcDNA3) or plasmid encoding C3aR using calcium phosphate (32) and were used after 48–60 h of culture. Culture of mouse bone marrow-derived macrophages Bone marrow was harvested from both mouse femurs and tibias and single cell suspension was obtained by passing through a syringe, and the cells were plated into 15 × 100 mm petri dishes containing 10 ml of RPMI medium supplemented with 10% FCS, glutamine (2 mM), penicillin (100 U/ml), streptomycin (100 µg/ml), and 15% L929 cell condition medium (LCM) and cultured for 2 days. Nonadherent cell fraction was collected and cultured for an additional 5 days. Once the cells reached confluence, cell purity was checked by FACS analysis using a macrophage marker antibody, F4/80 (Research Diagnostic). RT-PCR of C3aR and C5aR Total RNA was isolated from cells using TRIzol Reagent (Life Technologies, Rockville, MD) according to the manufacturer’s instructions. Digestion of any contaminating DNA was achieved by treatment of samples with RQ1 RNase-free DNase. Reverse transcription was performed with 5 µg of RNA using the Superscript II RNase H-Reverse Transcriptase (Invitrogen) according to the manufacturer’s protocol. PCR was then performed with the following primers for human C3aR: forward primer 5′-CGC GAA ATC TTC ACT ACA GAC AAC C-3′ and reverse primer 5′-TCA CCT AGT GAT CGT TAT TGC CAC GA-3′. The primers for the human C5aR: forward primer 5′-GAG CCC AGG AGA CCA GAA CAT G -3′ and reverse primer 5′-TAC ATG TTG AGC AGG ATG AGG GA -3′. The primers for the human β-actin: forward primer 5′ -AAG GCC AAC CGC GAG AAG ATG A-3′ and reverse primer 5′-GGA AGA GTG CCT


CAG GGC AGC G-3′. The primers for the mouse C3aR were forward primer 5′-CTC ACT TGT CTA TTG GGA CTG CTA-3′ and reverse primer 5′-AGC TGA GCA GTG GAG TTA TCA GTA G-3′. The primers for the mouse C5aR were forward primer 5′-CCT TAT CAT CTA CTC GGT GGT GTT-3′ and reverse primer 5′-CAC ATA CAG TGT GCT CTG AGT AGA AGT C3′. The primers for the mouse β-actin were forward primer 5′-TCA TGA AGT GTG ACG TTG ACA TCC GT-3′ and reverse primer 5′-CTT AGA AGC ATT TGC GGT GCA CGA TG-3′. PCR products (15 µl) were separated by electrophoresis in 1.2% agarose gels. Flow cytometry The expression of cell surface C3aR and SCF were determined by flow cytometry as previously described (33). Briefly, 0.25 × 106 cells were washed with HEPES-buffered saline containing 0.1% BSA and incubated with rabbit IgG or rabbit anti-human C3aR antibody or anti-human SCF antibody (10 µg/ml) for 1 h at 4°C, washed, and subsequently incubated with FITC- or PElabeled goat anti-rabbit IgG secondary antibody. After washing twice, cells were fixed in 1% paraformaldehyde and analyzed on a FACStarPLUS flow cytometer (BD Biosciences, Mountain View, CA). Confocal microscopy Human ASM cells were cultured on coverslips and transfected with either control vector (pcDNA3) or plasmid encoding C3aR and grown for 60 h. ASM cells (on coverslips) and HMC1 cells (in suspension) were washed with PBS containing 0.1% BSA, fixed with 4% paraformaldehyde for 30 min, and blocked with PBS containing 0.1% BSA for 30 min. Cells were then incubated with either rabbit IgG or rabbit anti-human C3aR antibody (10 µg/ml) for 1 h at room temperature. Cells were washed and incubated with goat anti-rabbit secondary antibody conjugated to FITC (1:200 dilution) for 1 h at room temperature. Cells were washed again and stained with DAPI (10 µM) for 30 min. Coverslips were then mounted on slides with antifade mounting solution (Molecular Probes) and sealed. Fluorescence was imaged by confocal microscopy (Bio-Rad [Temecula, CA] Radiance 2100) using the blue diode (excitation λ=405) and argon (excitation λ=488) lasers. Fluorescent emissions were captured using filter 476 ± 48 for DAPI and 515 ± 30 for FITC. Immunohistochemical analysis Dual immunostaining of human trachea and bronchus tissue sections were performed as described previously (34). Briefly, specimens were fixed with 3.7% paraformaldehyde for 15 min at room temperature and sections were blocked with 0.5% TSA Fluorescein System blocking reagent (NEN Dupont, Boston, MA) in 20 mM Tris (pH 7.5) and 150 mM NaCl (TBS) for 1 h at 37°C. Next, incubation with primary anti-human C3aR antibody at 1:100 dilution for 1 h at 37°C, and then secondary antibodies Alexa Fluor 594 anti-rabbit IgG conjugate, at 1:400 dilution for 1 h at 37°C, were performed. After incubation with anti-smooth muscle α-actin clone 1A4 FITC conjugate antibody, the sections were mounted in Vectashield mounting medium (Vector Laboratories, Burlingame, CA). The immunostaining was visualized on Nikon Eclipse TE2000-E Inverted Microscope (Nikon Instruments, Melville, NY) equipped with Evolution QEi digital video camera (Media Cybernetics, Silver Spring, MD) under ×200. Negative controls


included omission of the primary antibody and replacement of the primary antibody with rabbit IgG. Calcium measurements Ca2+ mobilization was determined as described previously. Briefly, cells (1×106) were loaded with 1 µM indo-1 AM in the presence of 1 µM pluronic F-127 for 30 min at room temperature. Cells were washed and resuspended in 1.5 ml of HEPES-buffered saline (35). Ca2+ mobilization was measured in a Hitachi F-2500 spectrophotometer with an excitation wavelength of 355 nm and an emission wavelength of 410 nm, and the data were expressed as a ratio of 355/410. Measurement of interleukin-8 (IL-8) secretion by ASM cells Confluent ASM cells were growth arrested by incubating the monolayers in Ham’s F12 with 0.1% bovine serum albumin (BSA) for 24 h. Cells were then exposed to LPS or IL-1β for an additional 24 h. IL-8 secreted in the culture supernatant was measured by ELISA as described previously (36). Assay of degranulation in mast cells For studies on mast cell degranulation, a recently described mast cell line, LAD 2, was used. This cell line was developed from the bone marrow of a patient with mast cell leukemia (37). LAD 2 mast cells were maintained in StemPro-34 medium (Life Technologies, Grand Island, NY) with 100 ng/ml stem cell factor (37). For degranulation, mast cells (5×103) were incubated with or without monolayer cultures of ASM cells (5×104) or culture supernatants in 24-well plates for indicated time periods in a total volume of 150 µl of buffer containing 0.1% BSA (35). Cells were then exposed to C3a (10 nM) for 30 min, and the extent of degranulation was determined by measuring the release of β-hexosaminidase. The data are presented as percent release (35). RESULTS Mast cells and monocyte/macrophages express C3a and C5a receptors, but ASM cells do not C3aR and C5aR are endogenously expressed in human mast cells and circulating leukocytes (36, 38–41). Whether C3a and C5a receptors are expressed in cultured primary human and murine ASM cells remains unknown. Initially, 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 and purified human monocytes, which constitutively express C3aR and C5aR, served as a control. As shown in Fig. 1A, C3aR and C5aR mRNA was expressed by HMC-1 cells and human monocytes. Surprisingly, we found that mRNA for these receptors were undetectable in human ASM cells. Flow cytometric analysis using C3aR-specific antibody also demonstrated the presence of C3aR on the surface of mast cells but not ASM cells (data not shown). C3a and C5a mediate their biological response via the activation of G protein-coupled receptors that activate phospholipase Cβ and mobilize intracellular Ca2+ (36). Ca2+ mobilization is a


sensitive assay measuring receptor expression and function. Using this assay, we confirmed the functional expression of C3aR and C5aR in different cells. We found that both C3a and C5a caused Ca2+ mobilization in HMC-1 cells (Fig. 1B). The anaphylatoxins, however, did not stimulate Ca2+ responses in human ASM cells (Fig. 1C). For control, we tested the effect of bradykinin, which activates Gq protein-coupled receptors on human ASM cells, to stimulate Ca2+ mobilization (42, 43). As shown in Fig. 1C, ASM cells that were unresponsive to C3a or C5a for Ca2+ mobilization responded robustly to bradykinin. 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 (12, 22, 44). We therefore used RT-PCR to assess the expression of C3aR and C5aR mRNA in primary cultures of murine ASM cells. For comparison, bone marrow-derived macrophages and an alveolar macrophage cell line MH-S were also used. As shown in Fig. 1D, macrophages expressed both C3aR and C5aR mRNA. In contrast, 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 (Fig. 1E). In contrast, C3a did not stimulate Ca2+ mobilization in murine ASM cells despite the fact that bradykinin stimulated a robust Ca2+ response (Fig. 1F). Immunolocalization of C3aR expression in vitro and in vivo We used transfected ASM cells and confocal microscopy as an additional approach to determine C3aR expression on ASM cells. HMC-1 cells, which endogenously express C3aR, served as a control. Cells were incubated with C3aR antibody or rabbit IgG followed by FITC-conjugated anti-rabbit IgG antibody. Single cells were then analyzed for C3aR expression by confocal microscopy. C3aR could be detected on the surface of HMC-1 (Fig. 2B) but not on ASM cells (Fig. 2D). To confirm that human ASM cells do not endogenously express cell surface C3aR, we generated transient transfectants with cDNA encoding this receptor. The transfected cells were then stained as described above, and cell surface expression of C3aR was determined by confocal microscopy. C3aR was readily detected on the surface of transfected ASM cells (Fig. 2F). ASM cells respond to proinflammatory mediators (LPS) and cytokines such as IL-1β for transcription factor activation and cytokine production (45, 46). This raises the possibility that C3aR could be expressed in ASM cells de novo upon stimulation with inflammatory mediators and cytokines. To test this possibility, we cultured ASM cells with LPS (100 ng/ml) or IL-1β (10 ng/ml) for 24 h and assayed for the production of proinflammatory cytokines in culture supernatants and C3aR expression on cell surface. We found that unstimulated ASM cells produced 32.2 ± 5 pg/ml of IL-8 in culture supernatant. LPS and IL-1β 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. Furthermore, C3a failed to induce intracellular Ca2+ mobilization in LPS- or IL-1β-treated ASM cells (data not shown). 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. To examine whether or not correlation exists between C3aR expression in vivo and in vitro, we performed immunohistochemical analysis of C3aR expression in human trachea and bronchus.


Smooth muscle-positive cells in human bronchus (Fig. 3A) and trachea (Fig. 3B) have little immunoreactivity for C3aR. In contrast, cells in the epithelial layer show marked immunostaining for C3aR as compared with tissue sections stained with isotope-matched IgG (Fig. 3A, upper panel). These 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 ASM is not associated with loss of C3aR expression due to cell culture. Human ASM cells enhance C3a-induced mast cell degranulation Asthmatic patients have increased numbers of mast cells in the smooth muscle layer of airways as compared with normal subjects (47). Furthermore, degranulated mast cells are detected in greater number in ASM of patients who died from asthma when compared with nonasthmatic control (10). These findings raise the interesting possibility that interaction of ASM cells with mast cells could lead to an enhancement of C3a-induced degranulation. HMC-1 cells thus far used in the present studies are from an immature mast cell line, and although it expresses C3aR, activation of this receptor does not lead to mediator release (36). To test the role of ASM cells on the regulation of C3aR function in mast cells, we used a recently characterized mast cell line, LAD 2 (37, 48), that displays characteristics of mature mast cells. We found that a low concentration of C3a (10 nM) caused robust mast cell degranulation (Fig. 4A). Furthermore, coculture of mast cells with ASM cells for 24 h resulted in a significant enhancement of C3ainduced response. To test the possibility that the factors released from ASM cells enhance C3ainduced mast cell degranulation, we also incubated mast cells for 24 h with ASM culture supernatant. As shown in Fig. 4A, in contrast to ASM cells, its culture supernatant had no effect on C3a-induced mast cell degranulation. Furthermore, coculture of ASM cells and mast cells for 15 min was sufficient to enhance C3a-induced mast cell degranulation (Fig. 4B). These findings suggest that rapid cell-cell contact between mast cells and ASM cells is required for enhanced C3a-induced mast cell mediator release. 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 and it also regulates their growth, function, and survival (49, 50). Kassel et al. (51) recently showed that ASM cell is an important and constitutive source of SCF and is expressed both as membrane-bound and soluble forms. Using flow cytometry, we confirmed that SCF is present on the surface of ASM cells (Fig. 5A) but not mast cells (Fig. 5B). This suggests that ASM-bound SCF could interact with its receptors c-kit on mast cells to enhance C3a-induced degranulation. To test this possibility, we first preincubated SCF (20 ng/ml) with its neutralizing antibody (5 µg/ml, 1 h), and mast cells with a neutralizing antibody to c-kit (5 µg/ml) (52) and tested their effects on SCF-induced Ca2+ mobilization. As shown in Fig. 5C, these antibodies completely blocked SCF-induced Ca2+ mobilization in mast cells. However, when ASM cells and mast cells were preincubated with anti-SCF and anti-c-kit antibodies, respectively, even at concentrations of 10 µg/ml, they had no effect on C3a-induced mast cell degranulation or the enhancement by ASM cells (Fig. 5D). To test the role of SCF on C3a-induced mast cell degranulation further, we stimulated mast cells with C3a (10 nM) in the absence or presence of recombinant human SCF (10–100 ng/ml) and determined mast cell degranulation. We found that SCF had no effect on C3a-induced mast cell degranulation (data not shown).


Dexamethasone-treated ASM cells are less effective than untreated cells in enhancing C3ainduced mast cell degranulation The therapeutic actions of glucocorticoids in asthma appears to be mediated via their effects on ASM cells and mast cells (53, 54). Dexamethasone regulates the expression of cell surface adhesion molecules on ASM cells (53, 55). We therefore treated ASM cells with dexamethasone (10 and 100 nM for 16 h) and tested its effects on mast cell function. As shown in Fig. 6A, dexamethasone treatment of ASM cells had no effect on the expression cell surface SCF. However, steroid-treated ASM cells were significantly less effective than untreated cells in enhancing C3a-induced mast cell degranulation (Fig. 6B). Treatment of mast cells with dexamethasone (10 nM for 1 h) had no effect on C3a-induced mast cell degranulation (data not shown). These findings suggest that the inhibitory effect of dexamethasone on mast cell/ASM interaction is due to its action on ASM and not mast cells. DISCUSSION This study demonstrates the surprising observation that while mast cells express functional C3aR and C5aR, human and murine ASM cells do not. It also reveals the novel findings that cell-cell interaction between mast cells and ASM cells enhances C3a-induced mast cell degranulation, thus providing a possible mechanism for the role of anaphylatoxins in the pathogenesis of allergic asthma. Cultured primary human ASM cells have been used extensively to study signaling pathways and biological responses mediated by cytokines and ligands for G protein-coupled receptors (11, 56, 57). Using RT-PCR, we found that steady-state mRNA levels for C3aR and C5aR were expressed in mast cells but not in human ASM cells. Furthermore, flow cytometry analysis showed that C3aR was not expressed on human ASM cells. The finding that C3aR was detected on human ASM cells only when transfected with C3aR supports the notion that these cells do not endogenously express C3aR. At a functional level, we found that C3a and C5a mobilized cytosolic Ca2+ in mast cells but had no effect on human ASM cells. These findings suggest that C3a plays a role in asthma via the activation of its receptors in mast cells but not ASM cells. Studies in certain rodent models indicated that the effects of C3a in experimental asthma does not involve leukocyte recruitment and presumably occurs in the absence of mast cell activation (12, 21, 22). It is therefore possible that species-specific differences exist in the mechanism by which C3a promotes AHR. Thus, mast cell activation and leukocyte recruitment could play a major role in human disease while ASM cells participate in experimental asthma in rodents. If this were the case, one would expect differences in the expression of C3aR and C5aR in human vs. murine ASM cells. However, we found that cultured primary murine ASM cells did not express C3aR or C5aR, although bone marrow-derived macrophages and an alveolar macrophage cell line did. Furthermore, both C3a and C5a stimulated intracellular Ca2+ mobilization in macrophages whereas C3a did not induce such responses despite the fact that bradykinin was an effective agonist. These findings, therefore, contradict the view that the effects of anaphylatoxins in asthma are mediated via the direct activation of their receptors on ASM cells (12, 21, 22, 44, 58).


The finding in the present study that human ASM cells do not express anaphylatoxin receptors is in agreement with studies by Fayyazi et al. (59, 60) but contradicts those of Drouin et al. (22). Using immunohistochemistry and in situ hybridization in paraffin-embedded tissue, Drouin et al. (22) have shown that C3aR are expressed on bronchiolar epithelial cells. They also detected C3aR on the basolateral surface of bronchioles. Immunohistochemistry analysis of separate sections demonstrated the presence of α-actin on the basolateral surface of bronchioles. Based on these findings, it was proposed that C3aR is expressed on both epithelial and ASM cells in the bronchiole. The data presented in Fig. 3 are in agreement with Drouin et al. regarding the expression of C3aR in airway epithelial cells but not ASM cells. Using double-labeled lung tissue, we demonstrated that while C3aR is expressed abundantly in airway epithelial cells, it is absent in ASM cells. The reason for the difference between our studies and those of Drouin et al. (22), with respect to C3aR in ASM cells in vivo, is not clear but could reflect the sensitivity of the staining procedures used and the utilization of double labeling and colocalization as opposed to single staining. Increased levels of C3a are detected in BAL fluids following allergic challenge (12, 13). Furthermore, C3a is one of the most potent mast cell chemoattractants known (14, 61), and it also induces mast cell degranulation (48, 62). This raises the possibility that the effects of C3a in asthma reflect, at least in part, the recruitment and activation of mast cells or other inflammatory cells into the airway. This view is supported by the following observations. First, C3a does not cause contraction of isolated murine tracheal strips and it also fails to induce AHR or airway inflammation after intratracheal instillation in naïve mice (58, 63). In contrast, in mice immunized with house dust mite, subsequent intratracheal administration of C3a induces both AHR and airway inflammation (58). Second, mast cell-deficient mice do not develop AHR following exposure to aerosolized allergen in the absence of adjuvant (64). Furthermore, 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 following allergen exposure. Third, studies with human subjects have also provided evidence for a close interaction between mast cells and ASM cells in asthma (29, 47, 65). Thus, immunohistological analysis of biopsy specimens from subjects with asthma revealed a striking increase in the number of mast cells among ASM bundles when compared with those from normal subjects (8, 9). Furthermore, degranulated mast cells are detected in greater numbers in ASM of patients who died from asthma when compared with nonasthmatic control (10). The novel and most interesting finding of the present study is that coculture of mast cells with ASM cells resulted in an enhancement of C3a-induced mast cell degranulation. This suggests that C3a-induced release of mast cell mediators contracts ASM. In addition, the ability of ASM cells to enhance mast cell degranulation further enhances smooth muscle contraction. However, the mechanism by which ASM cells enhance C3a-induced mast cell degranulation is not known. 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 is required for this type of cross-regulation. Yamamoto et al. (52) showed that mast cells interact with fibroblast via SCF and c-kit. However, the demonstration in the present study 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 were significantly less effective in enhancing C3a-


induced mast cell degranulation when compared with untreated cells. Human ASM cells and mast cells express a variety of adhesion molecules such as vascular cell adhesion molecule-1, intracellular adhesion molecule-1, and their counter receptors (65, 66). Mast cells also express a novel adhesion molecule, spermatogenic immunoglobulin superfamily (SgIgSF) (67). Whether these or other molecules on ASM cells and mast cells interact to enhance C3a-induced mast cell degranulation remains to be determined. In conclusion, we made the novel observation that ASM cells enhance C3a-induced mast cell degranulation following cell-cell contact. This finding not only strengthens the role of C3a in the pathogenesis of asthma, it provides a possible mechanism by which it regulates AHR. It suggests that C3a-induced release of mast cell mediators contracts ASM. In addition, the ability of ASM cells to enhance mast cell degranulation further enhances smooth muscle contraction. The demonstration that dexamethasone-treated ASM cells were significantly less efficient than untreated cells in enhancing C3a-induced mast cell degranulation suggests that C3a receptors on mast cells could be exploited as therapeutic targets in diseases characterized by airway inflammation. ACKNOWLEDGMENTS We are grateful to Drs. Arnold Kirshenbaum and Dean Metcalfe (NIAID/NIH) for LAD 2 mast cell line. We also thank Dr. Joseph Butterfield (Mayo Clinic, Rochester, MN) and Center for AIDS Research Immunology Core (University of Pennsylvania) for HMC-1 cells and human monocytes, respectively. This work was supported by National Institutes of Health grants 1RO1HL63372 (HA), 2R01-HL-55301, HL67663 (RAP), and 2RO1-HL64063 (YA). Yassine Amrani is a Parker B. Francis Fellow in Pulmonary Research. REFERENCES 1.

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Fig. 1

Figure 1. Mast cells and monocyte/macrophages express C3a and C5a receptors, but ASM cells do not. A) RT-PCR analysis of C3aR, C5aR, and β-actin mRNA in a human mast cell line HMC-1 (lane 1), human monocytes (lane 2), and human ASM cell (lane 3). B) HMC-1 cells were loaded with indo-1 AM and stimulated with C3a (10 nM) and C5a (10 nM), and Ca2+ mobilization was determined. C) Human ASM cells were also loaded with indo-1 AM and stimulated with C3a (10 nM), C5a (10 nM), and bradykinin (BK, 5 µM) as indicated, and Ca2+ mobilization was determined. D) RT-PCR analysis of C3aR, C5aR, and β-actin mRNA in murine bone-marrow-derived macrophages (lane 1), an alveolar macrophage cell line, MH-S (lane 2), and murine ASM cells (lane 3). E) Bone-marrow-derived macrophages were loaded with indo-1 AM and exposed to C3a (10 nM) and C5a (10 nM) as indicated by the arrows, and Ca2+ mobilization was determined. F) Murine ASM cells were also loaded with indo-1 AM and Ca2+ mobilization in response to C3a (10 nM), and bradykinin (BK, 5 µM) was determined. Data shown are representative of three similar experiments. Page 17 of 22 (page number not for citation purposes)

Fig. 2

Figure 2. Immunolocalization of C3aR in HMC-1 and human ASM cells by confocal microscopy. HMC-1 cells (A, B) and untransfected human ASM cells (C, D) were incubated with control antibody (A, C) or anti-human C3aR antibody (B, D) followed by FITC-conjugated anti-rabbit IgG antibody. Human ASM cells were transfected either with control vector (E) or C3aR (F). Cells were incubated with anti-human C3aR antibody followed by FITC-conjugated anti-rabbit IgG antibody. Cells were incubated with DAPI (to stain nucleus), and cell surface receptor expression was determined by confocal microscopy. Page 18 of 22 (page number not for citation purposes)

Fig. 3

Figure 3. Smooth muscle positive cells in human bronchus (A) and trachea (B) show no immunoreactivity for C3aR. Tissue sections were immunostained with anti-human C3aR antibody (red), and then secondary antibodies Alexa Fluor 594 anti-rabbit IgG conjugate, followed by immunostaining with anti-smooth muscle α-actin-FITC-conjugated (SM α-actin, green) mouse monoclonal antibody. ASM, airway smooth muscle; E, epithelium. Tissue specimens from two donors were analyzed on Nikon Eclipse TE2000-E microscope at ×200. The image shown is representative of two separate experiments. Images 1–3 show enlarged insets.


Fig. 4

Figure 4. Human ASM cells enhance C3a-induced mast cell degranulation. A) Mast cells were cultured with medium, ASM cells, or ASM cell-culture supernatant for 24 h. Cells were then stimulated with or without C3a (10 nM, 30 min), and the extent of degranulation (β-hexosaminidase release) was determined. Data are presented as percent β-hexosaminidase release. B) Mast cells were preincubated with ASM cells for different time periods and stimulated with C3a (10 nM, 30 min), and percent β-hexosaminidase release was determined. The data are the mean ± SE of four experiments performed in triplicate (***P