Monocyte Chemoattractant Protein-1 Mediates Cockroach Allergen-Induced Bronchial Hyperreactivity in Normal But Not CCR2^−/− Mice: The Role of Mast Cells¹

Mechanisms that confer the selective migration and activation of leukocytes during an inflammatory response have been widely investigated. The chemokines, a family of 8- to 10-kDa proteins, have been postulated to be pivotal through their ability to attract distinct cell types into various inflammatory lesions (1, 2, 3, 4, 5). In addition to their ability to induce movement of cells, recent data have demonstrated that chemokines are involved in cellular activation/degranulation (6, 7, 8). The biology of these mediators, however, appears to be associated with some level of redundancy because most chemokine receptors bind multiple ligands and most cells express numerous receptors. Nevertheless, selective leukocyte activation in the CC chemokine family may have important therapeutic implications in allergic airway inflammation in which the predominant infiltrates are eosinophils, lymphocytes, and monocytes (9, 10, 11, 12, 13, 14). Specifically, eotaxin, a potent activator of eosinophils (14, 15, 16, 17, 18) and Th2 lymphocytes (19), interacts with CCR3, whereas CCR2 receptor is selective for the monocyte chemoattractant proteins (20, 21, 22, 23, 24, 25, 26).

A number of studies have linked the induction of airway reactivity in atopic asthma with the presence of mononuclear cell and eosinophilic inflammation in the lung. Earlier work from our group demonstrated that inhibition of monocyte chemotactic protein-1 (MCP-1)³ in a murine model of Schistosoma mansoni egg Ag-induced allergic airway inflammation attenuated the associated mononuclear cell infiltration and airway hyperreactivity (27). MCP-1 is not a potent eosinophil chemoattractant but has been shown to be an important mediator of monocyte and CD4⁺/CD8⁺ lymphocyte recruitment (28, 29, 30). In addition, this chemokine also is a potent histamine-releasing agent for basophils and mast cells (31, 32, 33, 34). In this study, we have investigated the relationship between MCP-1 and the induction of bronchial hyperreactivity using a cockroach Ag-induced model of allergic airway disease. We present evidence that MCP-1 is critically involved in the induction of changes in airway resistance in allergic and normal mice through the activation of local mast cell populations.

Female CBA/J mice were purchased from The Jackson Laboratory (Bar Harbor, ME) and were maintained under standard pathogen-free conditions. CCR2-targeted mice (50:50 129SV:C57BL6) (35) were bred in the University of Michigan (Ann Arbor, MI) animal facilities.

Airway hyperreactivity was measured using a Buxco mouse plethysmograph, which is specifically designed for the low tidal volumes (Buxco, Troy, NY) as previously described (27). Briefly, the mouse to be tested was anesthetized with sodium pentobarbital and intubated via cannulation of the trachea with an 18-gauge metal tube. The mouse was subsequently ventilated with a Harvard pump ventilator (tidal volume, 0.4 ml; frequency, 120 breaths/min; positive end-expiratory pressure, 2.5–3.0 cm H₂O), and the tail vein was cannulated with a 2-gauge needle for injection of the methacholine challenge. The plethysmograph was sealed, and readings were monitored by computer. Because the box was a closed system, a change in lung volume was represented by a change in box pressure (P_box) which was measured by a differential transducer. The system was calibrated with a syringe that delivered a known volume of 2 ml. A second transducer was used to measure the pressure swings at the opening of the trachea tube (P_aw), referenced to the body box (i.e., pleural pressure), and to provide a measure of transpulmonary pressure (P_tp = P_aw − P_box). The trachea transducer was calibrated at a constant pressure of 20 cm H₂O. Resistance is calculated by the Buxco software by dividing the change in pressure (P_tp) by the change in flow (F) (δP_tp/δF; units = centimeters H₂O/ml/s) at two time points from the volume curve based on a percentage of the inspiratory volume. The mouse was attached to the box and ventilated for 5 min before acquiring readings. Once baseline levels were stabilized and initial readings were taken, a methacholine challenge was given via the cannulated tail vein. After determination of a dose-response curve (0.001 to 0.5 mg), an optimal dose was chosen, 0.1 mg of methacholine. This dose was used throughout the rest of the experiments in this study. After the methacholine challenge, the response was monitored, and for each animal the peak airway resistance minus the baseline resistance was recorded as a measure of change in airway reactivity. Data are means ± SE peak change in airway resistance (centimeters H₂O/ml/s) for n animals.

Normal CBA/J mice were sensitized and challenged with cockroach Ag to induce a Th2-type response. Briefly, mice were immunized i.p. with 10 μg cockroach allergen (Bayer Corporation, Elkhart, IN) in IFA on day 0. On day 14, the mice were given an intranasal challenge of 10 μg cockroach allergen in 10 μl diluent to localize the response to the airway. This initial intranasal challenge with Ag induced little cellular infiltrate into the lungs of the mice on histological examination. Mice were then rechallenged 6 days later by intratracheal administration of 10 μg cockroach allergen in 50 μl sterile PBS or with PBS alone (vehicle). In depletion studies, mice were pretreated i.p. with polyclonal anti-murine MCP-1 (JE) Abs at 1 h before intratracheal challenge (10⁶/ml titer, 0.5 ml).

Normal CBA/J mice were anesthetized with ketamine (Bayer), the trachea exposed and preholed with an 24-gauge needle before direct instillation of 100 ng (50 μl) endotoxin-free recombinant murine CC chemokines (R&D Systems, Rochester, MN) into the airways. The animals were allowed to recover before lung function assessment.

To assess migration of cells into the airway, we subjected the mice to a 1-ml bronchoalveolar lavage (BAL) with PBS (PBS) containing 25 mM EDTA at various time points postchallenge. The cells were then dispersed using a cytospin (Shandon Scientific, Runcorn, U.K.) and differentially stained with Wright-Giemsa stain. The cell types (mononuclear phagocytes, lymphocytes, neutrophils, and eosinophils) were expressed as a percentage based on 200 total cells counted/sample. Morphometric analysis of eosinophils was accomplished by examining 100 high power fields (×1000 magnification) in histological sections from each lung.

Murine bone marrow cells were removed from isolated femurs by cannulating one end with a 26-gauge needle and washing with 2 ml of DMEM containing 1 mM d-glutamic acid, 10 mM HEPES, antibiotics, and 10% FCS with 10% rat T cell-stimulated culture supplement (Collaborative Biomedical Products, Bedford, MA). The cells were cultured in the above medium, supplemented with 0.1 ng/ml murine stem cell factor (SCF, Sigma) for 2–3 wk and passaged every 3 days. Pulmonary mast cells were isolated from the upper airways by cutting the tissue into ∼3-mm³ pieces and gently teasing apart with fine forceps before being cultured in medium containing murine SCF in a manner similar to that for the bone marrow cells as previously described (36). Briefly, the pulmonary mast cells were >95% c-kit positive by flow cytometry and could be induced to release histamine using classical degranulating reagents, such as 48/80 and SCF. Cells were stimulated at 1 × 10⁶/ml in DMEM without murine SCF for 1 h using recombinant murine chemokines (50 nM). As a positive control, some cells were sensitized with 1 μg/ml monoclonal anti-DNP-albumin IgE (Sigma) for 3 h, washed twice with medium, and incubated for 1 h with 100 ng/ml DNP-albumin.

The level of MCP-1 protein in whole lung homogenate was measured by specific ELISA with a modification of a double-ligand method as previously described (27). Briefly, lung tissue was homogenized on ice with a tissue tearer (Biospec Products, Racine, WI) for 30 s in 1 ml PBS containing 0.05% Triton X-100. The resulting supernatant was isolated after centrifugation (10,000 × g). To measure MCP-1 levels in this supernatant, flat-bottom 96-well microtiter plates (Nunc Immunoplate I 96-F, Roskilde, Denmark) were coated with 50 μl/well rabbit anti-MCP-1 polyclonal Abs for 16 h at 4°C and then washed with PBS and 0.05% Tween 20. Nonspecific binding sites were blocked with 2% BSA in PBS and incubated for 90 min at 37°C. Plates were rinsed four times with wash buffer, and cell-free supernatants were added (neat and 1/10) followed by an incubation for 1 h at 37°C. Plates were washed four times, streptavidin-peroxidase conjugate (Bio-Rad, Richmond, CA) was added, and the plates were incubated for 30 min at 37°C. Plates were washed again, and chromogen substrate (Bio-Rad) was added and incubated at room temperature to the desired extinction. The reaction was terminated with 50 μl/well 3 M H₂SO₄ solution, and the plates were read at 490 nm in an ELISA reader. Standards were 0.5-log dilutions of recombinant murine macrophage-inflammatory protein-1α (MIP-1α) or eotaxin from 1 pg/ml to 100 ng/ml. ELISAs for these chemokines did not cross-react with each other, MIP-1α, eotaxin, recombinant murine MCP-3, murine MCP-5, MIP-1β, MIP-2, KC IL-6, or murine TNF.

Cell-free lavage fluid or culture supernatants were immediately frozen before analysis by specific ELISA for histamine (Immunotech, Marseille, France) and/or LTC₄ (Caymen Chemical, Ann Arbor, MI) according to the manufacturers’ instructions, using a 1/3 dilution in PBS for BAL samples.

Statistical significance was determined by unpaired Student’s t test with p values < 0.05.

Sensitization and challenge with cockroach Ag induced time-dependent changes in airway resistance, peaking at 8 h and sustained at 24 and 48 h postchallenge (Fig. 1,A). Airway hyperreactivity did not follow the temporal accumulation of peribronchial eosinophilia, which increased 72 h postchallenge (Fig. 1,B). We have previously proposed that MCP-1, which does not have chemotactic activity for eosinophils, may be a key mediator in induction of airway hyperreactivity (27). Analysis of whole lung homogenates by specific ELISA revealed increases in chemokine levels for MCP-1, peaking at 8 h and declining by 24 h (Fig. 2). Similar patterns of protein expression were also observed for other CC chemokines, including eotaxin and MCP-3 (data not shown).

To specifically assess the role of MCP-1, sensitized mice were pretreated with anti-MCP-1 polyclonal Abs or normal rabbit serum (NRS) 1 h before challenge, and responses were monitored thereafter. Significantly less MCP-1 was detected in whole lung homogenates from animals pretreated with anti-MCP-1 Abs than in those from NRS controls 8 h postchallenge (0.65 ± 0.2 ng/ml and 1.48 ± 0.2 ng/ml, respectively, p = 0.02, n = 4 animals). Anti-MCP-1-treatment significantly attenuated airway hyperreactivity at 1 and 8 h, but not at 24 h post-allergen challenge (Fig. 3) compared with NRS time-matched controls. Thus, MCP-1 appears to play a critical role in the development of allergen-induced airway hyperreactivity.

Previous studies have demonstrated that MCP-1 can induce degranulation of mast cells (34). Analysis of sensitized CBA/J mice 8 h post-cockroach allergen challenge, the time of peak MCP-1 production, revealed that anti-MCP-1 Ab pretreatment reduced histamine levels in the BAL compared with NRS controls (Fig. 4). Because the most likely source of histamine in the mouse lung is from resident mast cells, cultures of isolated murine pulmonary and bone marrow mast cells were stimulated with recombinant murine MCP-1 (JE), and supernatants were analyzed at 1 h for histamine release. Murine MCP-1 (50 nM) induced degranulation of the pulmonary mast cells (Fig. 5,A) but had no effect on bone marrow-derived mast cells (Fig. 5 B) even though these cells clearly degranulated through IgE cross-linking. These latter results suggest that mast cell maturation may be relevant to MCP-1 responsiveness. In comparison, no significant mast cell histamine-releasing activity was observed from mast cells after stimulation with recombinant murine MCP-3, MIP-1α, or eotaxin, above vehicle control levels.

Because MCP-1 can directly induce mast cell activation and mast cells appear to be involved in asthma, we assessed the ability of these chemokines to influence airway resistance in the absence of airway inflammation. We instilled 100 ng murine MCP-1, MCP-3, MCP-5, eotaxin, MIP-1α, or vehicle into the lung of normal, nonsensitized CBA/J mice. At 1 h after administration, MCP-1-instilled mice demonstrated a significant increase in airway resistance (Fig. 6,A), which was dose dependent (Fig. 6 B). MCP-5 also appeared to induce hyperreactivity, although to a much lesser extent. Interestingly, both of these chemokines bind to and activate CCR2. In contrast, MCP-3, mMIP-1α, and eotaxin had no significant effect on lung function compared with vehicle-instilled animals. This ability of MCP-1 to induce a hyperreactive response in mice 1 h postinstillation was associated with increased levels of histamine in the BAL (57.1 ± 8.4 nM compared with 28.7 ± 4.5 nM from saline-instilled mice). Importantly, the ability of MCP-1 to induce hyperreactivity was prolonged, and 24 h postinstillation these animals demonstrated a significant increase (p < 0.01) in airway resistance of 23.8 ± 4.8 cm H₂O/ml/s, compared with a value of 5.2 ± 2.5 cm H₂O/ml/s for saline instillation. Although significant differences in the numbers of monocytes in the BAL were observed 8 h post-MCP-1 instillation compared with the saline controls (1.1 ± 0.14 × 10⁶ and 0.6 ± 0.2 × 10⁶, respectively; p < 0.02), the values between the treatment groups were not significantly different at 24 h.

To assess whether the effects of MCP-1 on hyperreactivity were mediated through the CCR2 receptor, which is the primary receptor for MCP-1, CCR2^−/− animals were sensitized and challenged with cockroach allergen. At the time of peak production of MCP-1, i.e., 8 h post-allergen challenge, CCR2^−/− mice demonstrated attenuated hyperreactivity compared with littermate controls (Fig. 7,A). Furthermore, the measurement of histamine levels in the CCR2^−/− mice demonstrated a significant decrease in BAL histamine levels compared with littermate control animals (Fig. 7 B). Likewise, mast cells from CCR2^−/− mice stimulated with MCP-1 demonstrated no histamine release over background, whereas those from littermate control mice had a 2.5-fold increase over background release levels (data not shown). In contrast, both CCR2^−/−- and CCR2^+/+-derived mast cells demonstrated similar IgE + Ag-stimulated histamine release. Interestingly, whole lung homogenates from the CCR2 knockout mice contained significantly higher levels of MCP-1 than their littermates post-allergen challenge (1.40 ± 0.2 ng/ml vs 0.66 ± 0.11ng/ml, respectively, p = 0.005, n = 6 mice), presumably reflecting the fact that the protein is not utilized by the CCR2 receptor. The CCR2^−/− animals demonstrated similar levels of peribronchial eosinophil accumulation compared with wild-type mice, at 24 h post-allergen challenge (648 ± 132 vs 765 ± 183 eosinophils/100 high power fields, respectively). Thus, airway hyperreactivity during an allergic response may be controlled through neutralization of MCP-1 or elimination of its receptor, CCR2.

In additional experiments, we also examined whether the CCR2^−/− mice had an attenuated response to direct instillation of MCP-1. Similar to the previous studies, we instilled 100 ng/ml MCP-1 intratracheally into naive animals and examined the mice at 24 h for induction of airway hyperreactivity. As depicted in Fig. 8, MCP-1 again induced airway hyperreactivity in littermate control mice that express CCR2. However, in the CCR2^−/− mice, a significant decrease in the airway hyperreactive response was observed. Altogether, these studies identify MCP-1 and CCR2 as critical components of the allergic airway responses that lead to altered physiological function.

Previous results have clearly demonstrated a role for leukotrienes in induction of prolonged changes in airway hyperreactivity in animal models. Because direct MCP-1 instillations induced prolonged hyperreactivity in nonallergic animals, we began to investigate whether MCP-1 directly induced the release of LTC₄ in the airways of mice. The results in Fig. 9 illustrate that in animals given MCP-1 down the airway, a significant increase of LTC₄ could be detected in the BAL fluid from the mice (Fig. 9,A). Likewise, when pulmonary mast cells were treated with MCP-1 in in vitro assays, a significant level of LTC₄ could be detected (Fig. 9,B). Finally, when we examined LTC₄ levels in allergic mice treated with anti-MCP-1, a significant decrease in LTC₄ levels compared with control Ab-treated animals was observed (Fig. 10). Altogether, these data suggest that MCP-1 initiates and maintains airway hyperreactivity in the airway partially through a leukotriene-mediated mechanism.

In this study, we present evidence that MCP-1 can induce the development of airway hyperreactivity and that it participates in the pathophysiology of allergic lung responses. The role for CC chemokine involvement in allergic airway disease has largely focused on the ability to attract eosinophils, T cells, and monocytes into the lung (37, 38, 39, 40, 41, 42, 43). The focus of much of the research in asthmatic inflammation has been on the role of eosinophils and their released products. In this study, we report that the inhibition of MCP-1, a chemokine that does not directly induce the chemotaxis of eosinophils, attenuates hyperreactivity in a cockroach Ag-induced model of allergic airway disease. Furthermore, the observation that direct instillation of MCP-1, but not MCP-3, MIP-1α or eotaxin, induces a bronchial hyperreactivity response in normal mice, suggests an alternative mechanism that does not directly involve the eosinophil. The additional evidence that the CCR2^−/− mice have an attenuated airway hyperreactive response during allergen challenge or after direct instillation of MCP-1 implicates a CCR2-mediated mechanism. Consistent with previous in vitro and in vivo data, the CCR2^−/− mice had a decreased mononuclear cell influx into the BAL with no apparent decrease in eosinophil accumulation. Therefore, MCP-1-mediated mechanisms were not associated with eosinophil biology.

Neutralization of MCP-1 during the allergic airway response decreased histamine in the BAL, whereas instillation of rMCP-1 into the airway of normal mice caused an increase in histamine release. Mast cell degranulation causes the release of a number of preformed mediators and those synthesized de novo (44). Our observations that MCP-1 is a potent histamine-releasing factor from pulmonary mast cells in vitro is consistent the work from other groups (31, 32, 33, 34). However, the ability of MCP-1 to activate pulmonary but not bone marrow mast cells may reflect important maturational differences between these two populations of mast cells and possibly the expression of CCR2 (studies in progress). Further studies in our laboratory indicate that isolated pulmonary murine mast cells constitutively express CCR2 mRNA (data not shown). The apparent disparity in the ability of ligands for this receptor that were tested in this study (MCP-1, -3, and -5) to induce hyperreactivity is not entirely clear but may reflect differing affinities at the level of the receptor or in the manner the ligand signals the mast cell. Alternatively, the ability to bind to multiple receptors, as in the case of MCP-3, may alter the CCR2 signaling and thus regulate the mast cell activation or induce an alternative signal pathway (45). It is interesting that increased levels of LTC₄ were measured in the BAL after instillation of MCP-1 into normal mice. The leukotrienes are potent, long lasting bronchoconstrictors (46), and their release may account for the longevity of the change in lung function observed after MCP-1 instillation. MCP-1 was originally identified as a monocyte chemoattractant and activating factor (47), and future studies will also examine the ability of MCP-1 to induce mediator and cytokine release by macrophages that ultimately influence airway function. Finally, we are investigating the possibility that MCP-1 might directly induce hyperreactivity via activation of smooth muscle cells surrounding the airways, because vascular smooth muscle expresses CCR2 (48). Whatever the mechanism, the differential effect is striking.

In our cockroach Ag model, we have observed that levels of MCP-1 are significantly elevated 8 h postchallenge, and it follows that the inhibition of hyperreactivity and histamine release in those mice pretreated with anti-MCP-1 Abs was maximal at this time point. Interestingly, Abs against MCP-1 attenuated the airway hyperreactivity at 1 h post-allergen challenge, even though MCP-1 levels were not elevated in whole lung homogenates at this time point. However, studies by Baghestanian et al. (49) indicate that MCP-1 is released from IgE-stimulated mast cells themselves, suggesting that local concentrations of this chemokine might be important very early in the allergic response. The primary source of MCP-1 during allergic inflammation is not clear, although a number of investigators have shown that the alveolar macrophage, pulmonary fibroblast, epithelium, and endothelium are also involved in its production (50, 51, 52, 53, 54, 55, 56). Several studies have now demonstrated that Th2-type cytokines (IL-4, IL-13) induce MCP-1 from multiple structural cell populations (57, 58) and may enhance mononuclear cell recruitment and mast cell activation in the allergic lung. In addition, eosinophils appear to be a significant source of MCP-1 (59). One can easily imagine a model in which activated eosinophils recruited to the airway interact and activate surrounding cell populations, including mast cells, via their ability to produce and release MCP-1.

The role of MCP-1 in human asthmatic responses is not entirely clear, however several studies have demonstrated that MCP-1 is up-regulated during these responses. The expression of MCP-1 protein in bronchial tissue from asthmatic compared with nonasthmatic patients was significantly increased, in that >50% vs <8% of the bronchial epithelial cells stained positive, respectively (52). In subsequent studies, investigators have demonstrated increased production of MCP-1 protein in allergic asthmatic patients compared with nonasthmatic subjects (38, 60). However, to date, few studies have attempted to identify whether MCP-1 has a detrimental role in allergic asthma. The fact that MCP-1 can induce LTC₄-mediated pathways make an important link between chemokine biology and arachidonic acid metabolites traditionally viewed as bronchoconstrictor mediators. The data from these studies may help to define a contributory role of MCP-1 in the exacerbation of allergic asthmatic responses and further suggest one possible mechanism of action within this response. Given the information provided in this and other studies, MCP-1 and CCR2 may represent suitable targets for therapeutic intervention of asthmatic responses. Altogether, these data outline how several mediator systems can be interrelated during the progression of allergic airway responses.

We thank Mary Glass for technical assistance.