Antigen-Induced Airway Hyperresponsiveness, Pulmonary Eosinophilia, and Chemokine Expression in B Cell Deficient Mice

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1 Antigen-Induced Airway Hyperresponsiveness, Pulmonary Eosinophilia, and Chemokine Expression in B Cell Deficient Mice James A. MacLean, Alain Sauty, Andrew D. Luster, Jeffrey M. Drazen, and George T. De Sanctis Clinical Immunology and Allergy Unit and Infectious Disease Unit, Department of Medicine, Massachusetts General Hospital; Pulmonary and Critical Care Divisions, Department of Medicine, Brigham and Women s Hospital; Partners Asthma Center; and Harvard Medical School, Boston, Massachusetts Murine models of allergen-induced pulmonary inflammation share many features with human asthma, including the development of antigen-induced pulmonary eosinophilia, airway hyperresponsiveness, antigen-specific cellular and antibody responses, the elaboration of Th2 cytokines (interleukin [IL]-4 and IL-5), and the expression of chemokines with activity for eosinophils. We examined the role of B cells and antigen-specific antibody responses in such a model by studying the histopathologic and physiologic responses of B cell deficient mice compared with wild-type controls, following systemic immunization and airway challenge with ovalbumin (OVA). Both OVA-challenged wild-type and B cell deficient mice developed (1) airway hyperresponsiveness, (2) pulmonary inflammation with activated T cells and eosinophils, (3) IL-4 and IL-5 secretion into the airway lumen, and (4) increased expression of the eosinophil active chemokines eotaxin and monocyte chemotactic protein-3. There were no significant differences in either the pathologic or physiologic responses in the B cell deficient mice compared with wild-type mice. These data indicate that B cells and antigen-specific antibodies are not required for the development of airway hyperresponsiveness, eosinophilic pulmonary inflammation, and chemokine expression in sensitized mice following aerosol challenge with antigen. MacLean, J. A., A. Sauty, A. D. Luster, J. M. Drazen, and G. T. De Sanctis Antigen-induced airway hyperresponsiveness, pulmonary eosinophilia, and chemokine expression in B cell deficient mice. Am. J. Respir. Cell Mol. Biol. 20: In murine models of allergen-induced pulmonary inflammation, CD4 T lymphocytes and the Th2 cytokines interleukin (IL)-4 and IL-5 are important regulators of eosinophilic pulmonary inflammation and airway hyperresponsiveness (1 8). The role of antigen-specific antibodies in these models is less clearly defined. Either immunoglobulin (Ig)E or IgG1 antigen-specific antibodies appear to be sufficient to induce pulmonary inflammation and airway hyperresponsiveness in immunized mice following antigen challenge (9, 10). However, whether the presence of antigenspecific antibodies is required to obtain all aspects of this asthma-like phenotype remains to be fully established. B cell deficient mice developed eosinophil-rich inflammation (Received in original form December 22, 1997 and in revised form May 27, 1998) Address correspondence to: James A. MacLean, M.D., Bulfinch 422, Massachusetts General Hospital, 50 Blossom St., Boston, MA maclean@helix.mgh.harvard.edu Abbreviations: bronchoalveolar lavage, BAL; BAL fluid, BALF; effective dose required to increase R L to 200% of control values, ED 200 R L ; enzymelinked immunosorbent assay, ELISA; fetal calf serum, FCS; immunoglobulin, Ig; interleukin, IL; monocyte chemotactic protein-3, MCP-3; ovalbumin, OVA; phosphate-buffered saline, PBS; pulmonary resistance, R L. Am. J. Respir. Cell Mol. Biol. Vol. 20, pp , 1999 Internet address: in the lung following systemic immunization and airway challenge with antigen (11, 12). However, B cell deficient mice sensitized via the airways did not develop airway hyperresponsiveness, despite the accumulation of eosinophils and Th2 cytokines in the bronchial tissue (12). Reconstitution of these B cell deficient mice with antigen-specific IgE resulted in the development of airway hyperresponsiveness, suggesting an important role for antigen-specific IgE in this process. In our own studies, we have found that in the absence of T cells (eliminated by anti-cd3 monoclonal antibody), the presence of antigen-specific IgE and IgG antibodies was not sufficient to induce pulmonary eosinophilia and the expression of the eosinophil-specific chemokine eotaxin in sensitized mice following antigen challenge (7). To evaluate further the role of antigen-specific antibody responses in the development of pulmonary eosinophilia, airway hyperresponsiveness, and chemokine expression in response to antigen, we evaluated B cell deficient mice following systemic immunization and repeated airway challenge with ovalbumin (OVA). Our findings demonstrate that airway hyperresponsiveness, pulmonary inflammation with activated T cells and eosinophils, and the expression of eosinophil-active chemokines can develop in the absence of B cells and antigen-specific antibody responses, to a degree that is comparable to that in wild-type mice.

2 380 AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL Materials and Methods Mice C57BL/6 wild-type mice ( / ) and C57BL/6-Igh-6 tm1cgn B cell deficient mice ( / ) (13) were obtained from The Jackson Laboratory (Bar Harbor, ME). The B cell deficient mice had been backcrossed onto the C57BL/6 strain for eight generations. The mice were studied between 7 and 10 wk of age. The mice were housed in a pathogen-free animal facility and were given food and water ad libitum. Immunization and Challenge Protocol Mice were immunized with 10 g of OVA (Sigma Chemical Co., St. Louis, MO) and 1 mg aluminum hydroxide intraperitoneally (i.p.) on Days 0, 7, and 14. Mice underwent aerosol challenge with either OVA (5% in phosphatebuffered saline [PBS]) or PBS for 20 min/d for 5 d, 7 d after the final immunization. Aerosol challenge was performed by placing mice in a Plexiglas box (dimensions: cm) and aerosolizing OVA using a DeVilbiss nebulizer (Somerset, PA) driven by compressed air. Mice were studied between 18 and 24 h after the last aerosol challenge. Pulmonary Resistance and Airway Responsiveness Airway responsiveness was measured as previously described (14, 15). In brief, dose-response curves to methacholine were obtained 18 to 24 h after the last aerosol challenge in anesthetized, ventilated mice. Methacholine was administered intravenously via the internal jugular vein, sequentially in increasing doses (33 to 1,000 g/kg). From the relationship between the dose administered and pulmonary resistance (R L ), without controlling for lung volume, the effective dose of methacholine that resulted in a doubling of R L was determined by log-linear interpolation. This dose is referred to as the effective dose required to increase R L to 200% of control values (ED 200 R L ) and was used as a measure of airway responsiveness. This index was log-transformed to reflect the log-linear relationship between the rise in resistance and the methacholine dose. Bronchoalveolar Lavage Bronchoalveolar lavage (BAL) was performed after the physiologic measurements, as previously described (7). In brief the lungs were lavaged with eight 0.5-ml aliquots of PBS containing 0.6 mm ethylenediamenetetraacetic acid (EDTA). Recovered live cells (trypan blue exclusion) were enumerated in a hemocytometer. Cell differential counts were determined by enumerating macrophages, neutrophils, eosinophils, and lymphocytes on Wright-stained (LEUKOSTAT; Fisher Scientific, Pittsburgh, PA) cytocentrifuge preparations of cells recovered by BAL. Antibodies Rat antimouse monoclonal antibodies used for tissue staining included: culture supernatants for anti-cd3 (clone YCD3), anti-cd4 (clone GK1.5), and anti-cd8 (clone ) raised in our laboratory. Commercial antibodies included anti-cd19, anti-b220, anti-cd11b (Mac-1), and anti-ly-6g (Gr-1) (all from PharMingen, San Diego, CA). Commercial conjugated antibodies used for flow cytometric analysis included anti-cd3-fitc (PharMingen), anti- CD4-PE (Becton Dickinson, San Jose, CA), anti-cd8- Red 613 (GIBCO BRL, Grand Island, NY), and anti- CD25 (PharMingen). Histology and Immunohistochemistry Lungs were harvested after BAL and either fixed in 10% formalin and embedded in paraffin blocks for regular histology, or embedded in cryopreservative and frozen for immunohistochemistry. Paraffin-embedded lung sections were stained with hematoxylin and eosin (H&E) using standard protocols. Frozen tissue sections were air-dried and fixed in acetone. The sections were blocked with normal serum from the species in which the biotinylated secondary antibody was raised (i.e., rabbit serum). Endogenous biotin was blocked using an Avidin-Biotin Blocking Kit (Vector Labs, Burlingame, CA). The sections were incubated with the primary monoclonal antibodies for 45 min, followed by washing in PBS. The sections were then incubated with the biotinylated secondary antibody for 45 min, followed by washing in PBS. The slides were developed using a standard avidin biotin enzyme complex method that uses alkaline phosphatase in the enzyme complex (Vectastain ABC-AP Kit; Vector), followed by the alkaline phosphatase substrate that produces a red reaction product. Cytokine Assays Quantification of IL-4 and IL-5 in BAL fluid (BALF) was performed by capture enzyme-linked immunosorbent assay (capture-elisa) kits according to the protocols provided by the manufacturers (IL-4/PharMingen; IL-5/Endogen, Cambridge, MA). The limit of detection of the IL-4 assay was 10 pg/ml, and for the IL-5 assay 5 pg/ml. RNA Extraction and Northern Blotting Lungs excised for RNA extraction were perfused with sterile saline via the pulmonary circulation to remove blood, and then frozen on dry ice and stored at 70 C before processing. Total cellular RNA was isolated from the lungs by homogenizing the tissue with a polytron in the presence of 4 M guanidine hydrochloride and pelleting the RNA through a 5.7 M CsCl 2 cushion (16). Total RNA, 15 g, was fractionated on a 1.2% agarose gel containing 0.2 M formaldehyde, transferred to GeneScreen (DuPont, Wilmington, DE), and hybridized with 32 P-[dCTP] Klenow-labeled random primed mouse eotaxin, mouse monocyte chemotactic protein-3 (MCP-3), or mouse ribosomal protein (rpl32) complementary DNA (cdna) probes. The ribosomal probe is a 279-base pair fragment encoding the ribosomal protein rpl32 and was used to control for RNA loading (17). Hybridization was performed in 50% formamide, 10% dextran sulfate, 5 saline sodium citrate (SSC), 1 Denhardt s solution (0.0002% [wt/vol] polyvinylpyrrolidone, % [wt/vol] bovine serum albumin, % [wt/vol] Ficoll 400), 100 g/ml of heat-denatured herring sperm DNA solution, 1% (wt/vol) sodium dodecyl sulfate (SDS), and 20 mm Tris (ph 7.5) at for 42 C for 18 h. Blots were washed with 2 SSC/0.1% SDS at 42 C for 40 min and then in 0.2 SSC/0.1% SDS at 55 C for 40 min. RNA was quantified using a PhosphorImager (Bio- Rad, Hercules, CA) and normalized to the signal for total RNA loaded (i.e., rpl32).

3 MacLean, Sauty, Luster, et al.: Antigen-Induced Pulmonary Inflammation in B Cell Deficient Mice 381 Flow Cytometry Flow cytometry was performed as previously described (18). In brief, cell suspensions were resuspended in staining buffer (PBS with 10% mouse serum) and stained with the conjugated antibodies at 4 C for 30 min. After washing with PBS, the cells were fixed with 1% paraformaldehyde. Single and three-color flow cytometry was performed on a FACScan cytofluorimeter (Becton Dickinson) and analysis was performed using Lysys software (Hewlett-Packard, Palo Alto, CA). OVA-Specific IgG and IgE Levels and Total Serum IgE OVA-specific IgG levels were measured as previously described (7). In brief, ELISA plates were coated with OVA and blocked with PBS 10% fetal calf serum (FCS) (Hazelton Biologicals, Inc., Lenexa, KS). Serum samples were added to the wells after serial dilution in PBS 10% FCS and were allowed to incubate for 1 h at room temperature. The plates were washed five times with PBS Tween before the addition of the secondary antibody, a goat antimouse IgG alkaline phosphatase (Vector), for 45 min at room temperature. The plates were washed 5 times with PBS Tween and 10 times with distilled water before development with phosphatase substrate (Kirkegaard & Perry Laboratories, Inc., Gaithersburg, MD). The reaction was allowed to proceed for 20 min and was stopped by the addition of EDTA. The plates were read in an ELISA plate reader at an optical density (OD) of 405 nm. OVA-specific IgE levels were measured by capture- ELISA as previously described (7). ELISA microtiter plates were coated with a purified antimouse IgE monoclonal antibody (Pharmingen) at a concentration of 2 g/ ml and blocked with PBS 10% FCS. Serum samples were diluted in PBS 10% FCS and incubated in the wells for 2 h. After washing with PBS Tween, biotinylated OVA (10 g/ml) was added to the wells and incubated for 1 h. The plates were washed with PBS Tween, followed by the addition of avidin alkaline phosphatase (Sigma) for 1 h. The plates were washed with PBS Tween and distilled water before the addition of the phosphatase substrate. The plates were allowed to develop for 30 min. The plates were read in an ELISA plate reader at an OD of 405 nm. Total serum IgE was measured by capture-elisa in a manner similar to the detection of OVA-specific IgE. A biotinylated rat antimouse IgE (Pharmingen) was used to detect captured IgE in place of the biotinylated OVA. Statistical Analysis Student s t test (unpaired, two-tailed) was used to calculate significance levels between treatment groups. Results Airway Responsiveness in OVA-Immunized/OVA- Challenged (OVA/OVA) Wild-Type and B Cell Deficient Mice Compared with OVA-Immunized/ PBS-Challenged (OVA/PBS) Control OVA/OVA wild-type mice had significantly increased methacholine responsiveness (as reflected by a lower log ED 200 R L ) compared with OVA/PBS wild-type control mice (P 0.01) (Figure 1). Similarly, OVA/OVA B cell deficient Figure 1. Airway responsiveness in OVA/PBS and OVA/OVA wild-type ( / ) and B cell deficient ( / ) mice. OVA/OVA wild-type ( / ) and B cell deficient ( / ) mice were significantly more responsive to methacholine challenge than were OVA/PBS-treated mice (P 0.01). There was no significant difference in responsiveness between the OVA/OVA ( / ) and ( / ) treated mice. Data are presented as means SEM of the ED 200 R L ; minimum of six mice per group. mice developed significantly enhanced methacholine responsiveness compared with OVA/PBS B cell deficient mice (Figure 1). There was no significant difference in airway responsiveness in OVA/OVA wild-type mice compared with OVA/OVA B cell deficient mice. These findings indicate that airway hyperresponsiveness develops in B cell deficient mice and that the increased responsiveness is not significantly different from that of similarly treated wildtype animals. Inflammatory Cell Recruitment and Th2 Cytokine Levels in the BAL of OVA/OVA Wild-Type and B Cell Deficient Mice The recovery of inflammatory cells from the airway lumen (BAL) and the harvesting of lung tissue were performed immediately following the physiologic assessments. There was no significant difference in BAL cell recoveries (total or specific cell types) between OVA/OVA wild-type and B cell deficient mice, indicating that B cell deficient mice can recruit inflammatory cells into the airway lumen as effectively as wild-type control mice can following antigen challenge. Recovery of cells from BAL of OVA/PBS wildtype and B cell deficient mice revealed a predominance of alveolar macrophages in both groups (Table 1). OVA/ OVA wild-type mice showed a significant increase in total cells, eosinophils, and lymphocytes compared with OVA/

4 382 AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL TABLE 1 BALF cell differential counts and cytokine levels Mouse Exposure Cell Count ( 10 6 cells) Differential Counts (% total cells) Macrophages Neutrophils Lymphocytes Eosinophils IL-5 (pg/ml) IL-4 (pg/ml) / OVA/PBS pg/ml 10 pg/ml / OVA/PBS pg/ml 10 pg/ml / OVA/OVA / OVA/OVA P value* P value P value NS NS NS NS NS NS NS Values are means SEM. NS not significantly different. *P value / OVA/OVA versus / OVA/PBS. P value / OVA/OVA versus / OVA/PBS. P value / OVA/OVA versus / OVA/OVA. PBS mice. OVA/OVA B cell deficient mice also demonstrated a significant increase in total cells, eosinophils, and lymphocytes recovered by BAL. Flow cytometric analysis of BAL cells demonstrated an increase of CD4 T cells bearing the activation marker CD25 (IL-2 receptor chain) in both the OVA/OVA B cell deficient mice and wild-type mice compared with the corresponding OVA/ PBS mice (data not shown). IL-4 and IL-5 levels were measured in the first milliliter of recovered BAL fluid from OVA/OVA wild-type and B cell deficient mice, and similar levels were found in both groups (Table 1). Levels were below the limit of detection in OVA/PBS-treated mice (Table 1). Interestingly, we noted that OVA/PBS B cell deficient mice had an increase in the percentage of lymphocytes and granulocytes in the BAL compared with similarly treated wild-type mice. The total cell counts in the OVA/PBStreated mice, however, were not different between wildtype and B cell deficient mice. Importantly, histologic analysis of the lungs of OVA/PBS B cell deficient mice did not show any evidence of pneumonia or pneumonitis (not shown). Lung Histology from OVA/OVA Wild-Type and B Cell Deficient Mice Histopathologic examination of lung tissue from OVA/ OVA wild-type mice revealed a pleomorphic peribronchial and perivascular infiltrate consisting of eosinophils, lymphocytes, macrophages, and neutrophils (Figure 2A) that was not seen in OVA/PBS mice (not shown). A similar histopathologic picture was seen in OVA/OVA B cell deficient mice (Figure 2B). Immunohistochemical staining of lung tissue demonstrated increased numbers of CD3 cells in the lungs of OVA/OVA wild-type (Figure 2C) and B cell deficient (Figure 2D) mice. CD4 and CD8 T lymphocytes, granulocytes (Gr-1 ), and monocyte/macrophages (Mac1 ) were also increased in both OVA/ OVA wild-type and B cell deficient mice (not shown). The only differential staining noted was in the number of B cells in the lungs of OVA/OVA wild-type mice (Figure 2E) that, as expected, were not identified in OVA/OVA B cell deficient mice (Figure 2F). Chemokine Messenger RNA (mrna) Expression in OVA/OVA Wild-Type and B Cell Deficient Mice Compared with OVA/PBS-Treated Control Mice The expression of eotaxin and MCP-3 mrna was assessed by Northern analysis of RNA recovered from whole-lung extracts, as previously described (7, 19). Eotaxin and MCP-3 mrna expression was induced in OVA/ OVA wild-type and B cell deficient mice compared with OVA/PBS-treated control animals (Figure 3A). Quantification of the mrna signal was achieved using a PhosphorImager (Figure 3B). There was a significant increase in eotaxin and MCP-3 mrna expression in OVA/OVA wild-type and B cell deficient mice compared with the respective OVA/PBS-treated mice. There was no significant difference in the expression of either eotaxin or MCP-3 between OVA/OVA wild-type and B cell deficient mice. Total IgE, OVA-Specific IgE, and OVA-Specific IgG in OVA/OVA Wild-Type and B Cell Deficient Mice Immunization with OVA was associated with a significant increase in total IgE, OVA-specific IgE, and OVA-specific IgG in wild-type mice (Figure 4). As expected, B cell deficient mice had undetectable levels of total IgE and did not develop measurable leveis of OVA-specific antibodies of either the IgE or IgG class (Figure 4). Discussion In B cell deficient mice, systemic immunization with antigen followed by repeated aerosol challenge resulted in the development of (1) airway hyperresponsiveness to methacholine, (2) pulmonary inflammation with activated T cells and eosinophils, (3) Th2 cytokine secretion in the lung, and (4) the induction of chemokines (eotaxin and MCP-3) with activity for eosinophils. The development of these parameters was not significantly different from similarly treated wild-type mice. These findings suggest that in the absence of B cells and antigen-specific antibody responses, the pathologic and physiologic changes that characterize this murine model can develop. Pulmonary inflammation with eosinophilia in systemically immunized and airway challenged B cell deficient mice has been previously reported; however, airway physi-

5 MacLean, Sauty, Luster, et al.: Antigen-Induced Pulmonary Inflammation in B Cell Deficient Mice 383 Figure 2. Lung histopathology and immunohistochemistry from OVA/OVA wild-type ( / ) and B cell deficient ( / ) mice. H&E stain of formalin-fixed lung tissue from OVA/OVA wild-type ( / ) (A) and B cell deficient ( / ) (B) mice showed a peribronchial and perivascular accumulation of inflammatory cells including eosinophils. Immunohistochemical staining revealed increased numbers of CD3 cells (red staining, arrows) in both OVA/OVA wild-type (C) and B cell deficient (D) mice that were not seen in OVA/PBS control animals (not shown). B cells (B220 ) were identified in the lungs of OVA/OVA wild-type mice (E) (red staining, arrows) but not in B cell deficient mice (F). Sections shown are from a representative experiment. Magnification: A D, 320; E and F, 200. ology was not examined by these investigators (11). In a separate study, Hamelmann and colleagues examined pathologic and physiologic lung responses in B cell deficient mice following repeated airway challenge (12). In this study, mice developed eosinophilic pulmonary inflammation, antigen-induced T cell-proliferation, and increased production of Th2 cytokines (IL-4 and IL-5) from bronchial lymph nodes stimulated with antigen in vitro. Interestingly, airway hyperresponsiveness was not seen in sensitized B cell deficient mice, but could be elicited following the passive transfer of antigen-specific IgE into immunized mice. These findings suggest that T cell responses to antigen were present in the B cell deficient mice but that airway hyperresponsiveness could develop only when antigen-specific IgE was present. In the current experiments, mice were immunized systemically (i.p.) and challenged by aerosol. This method of sensitization and challenge may account for our ability to

6 384 AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL Figure 3. Chemokine mrna expression in OVA/PBS- and OVA/OVA-treated wild-type ( / ) and B cell deficient ( / ) mice. RNA extracted from whole lungs 24 h after the last aerosol challenge was analyzed by Northern blotting. (A) The blot was sequentially hybridized with eotaxin, MCP-3 32 P-labeled cdna probes, and a probe for total RNA rpl32 (L32). (B) The RNA signal was quantified using a PhosphorImager and normalized to the rpl32 signal. Constitutive expression of eotaxin and MCP-3 mrna was seen in OVA/ PBS ( / ) and ( / ) mice. There was a significant increase in the expression of eotaxin in both the ( / ) and ( / ) OVA/OVA mice (*P and P 0.03, respectively). Similarly, there was a significant increase in the expression of MCP-3 in both the ( / ) and ( / ) OVA/OVA mice (both *P 0.03). There was no significant difference in the expression of eotaxin or MCP-3 between the OVA/OVA wild-type and B cell deficient mice. Each lane represents a different mouse. Data represent mean eotaxin or MCP-3 counts adjusted for RNA load SEM (OVA/PBS, three mice/group; OVA/OVA, minimum of five mice/group).

7 MacLean, Sauty, Luster, et al.: Antigen-Induced Pulmonary Inflammation in B Cell Deficient Mice 385 Figure 4. Total IgE, OVA-specific IgE, and OVA-specific IgG titers from OVA/PBS and OVA/OVA wild-type ( / ) and B cell deficient ( / ) mice. Serum obtained 24 h after the final aerosol challenge. Both OVA/PBS- and OVA/OVA-treated wild-type mice developed increased titers of total serum IgE (A), OVA-specific IgE (B), and OVA-specific IgG (C) compared with similarly treated B cell deficient mice (all *P 0.01). Data represent means SEM (minimum of four mice/group). OVA-spe- detect significant airway hyperresponsiveness in antigenchallanged B cell deficient mice in the absence of antigenspecific antibody responses. Takeda and associates have suggested that the protocol of sensitization and challenge used in any given murine model may determine the relative importance of antigen-specific antibodies and mast cells in regulating tissue eosinophilia and airway hyperresponsiveness (20). They showed that in the absence of mast cells, pulmonary eosinophilia and airway hyperresponsiveness developed after either repeated airway challenge or systemic sensitization followed by airway challenge (20). Similarly, eosinophilic pulmonary inflammation and airway hyperresponsiveness developed in IgE-deficient mice following repeated airway sensitization (21). In contrast, however, Kung and coworkers previously reported that mast cells may influence the degree of eosinophil recruitment to the lung following antigen challenge when the airway challenge was less intensive (22). As such, mast cells and IgE may play an important role in models in which sensitization and challenge are relatively attenuated. In contrast, T cells may play a more important role in models when the sensitization and challenge are repeated more frequently (23). This hypothesis is intriguing and may help explain the differences that have been noted between our findings and those of Hamelmann and associates (12). The relative importance of the route of sensitization in the development of airway hyperresponsiveness still remains to be fully established. Our findings suggest that the systemic method of sensitization leads to an increase in total IgE and in antigen-specific IgE and IgG antibodies in wild-type mice; however, the presence of these antibodies does not appear to act in a synergistic fashion to augment the histopathologic or physiologic changes in the model when compared with B cell deficient mice. Another factor that may have accounted for some of the differences between our studies in B cell deficient mice and those of Hamelmann and associates is the method used to measure airway hyperresponsiveness. We used an in vivo method that involves anesthetizing test animals and measuring airway responses to intravenously administered methacholine, a method we have found to be reproducible in the study of both immunized and naive animals (15, 24, 25). In contrast, Hamelmann and coworkers used an in vitro technique that has previously been demonstrated to correlate well with in vivo measurements (26). Finally, strain differences may have contributed to some of the differences between our study and that of Hamelmann and coworkers. They used B cell deficient backcrossed to B10.BR mice, whereas we used C57BL/6 B cell deficient mice. It has been suggested previously that airway hyperresponsiveness in C57BL/6 mice may be more dependent on cells other than mast cells because these mice are genetically deficient in important mast-cell mediators (e.g., mast-cell protease 7) (27). To the best of our current knowledge, genetic differences in airway hyperresponsiveness between B10.BR and C57BL/6 mice have not been established. cific IgG was assayed at a dilution of 1:500. Total IgE and OVAspecific IgE were assayed at a dilution of 1:3.

8 386 AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL lnterestingly, we noted that OVA/PBS-treated B cell deficient mice had an increased percentage of granulocytes and lymphocytes in the BALF, which was not seen in similarly treated wild-type mice. The total BALF cell counts, however, were not significantly different between these two groups, and we found no evidence of pneumonia or pneumonitis on gross lung histology in the B cell deficient OVA/PBS mice. The increased percentage of granulocytes and lymphocytes in the BALF of B cell deficient mice following PBS is likely related to the lack of antibodies in these mice. The increased percentage of lymphocytes and granulocytes in the BALF may reflect a compensatory mechanism for the lack of humoral immunity in the respiratory tract. Importantly, the increased percentage of lymphocytes and granulocytes in OVA/PBS B cell deficient mice did not alter the airway responsiveness compared with similarly treated wild-type mice. Furthermore, it did not interfere with the ability of these mice to respond to an antigenic challenge with the development of pulmonary inflammation with eosinophils and increased airway hyperresponsiveness. The accumulating evidence suggests that in murine models of allergen-induced airway hyperresponsiveness, the end points of airway hyperresponsiveness and pulmonary eosinophilia can develop by at least two distinct pathways: (1) a mast cell dependent pathway and (2) a CD4 T cell dependent pathway (23, 27). The mast cell dependent pathway is believed to result from the release of inflammatory mediators (e.g., tryptase, leukotrienes) from mast cells, which act on the airway microenvironment to increase responsiveness. Mast cell dependent airway hyperresponsiveness has been demonstrated both in the presence (12) and absence (28) of a sensitizing antigen or following the passive transfer of antigen-specific antibodies (9, 10). Other studies, however, clearly demonstrate that airway hyperresponsiveness can occur independently of IgE or mast cells (20, 21, 29). In these studies it is presumed that airway hyperresponsiveness results from the recruitment of eosinophils to the lung by antigen-specific T cells, with the subsequent release of eosinophil-derived inflammatory and cytotoxic mediators (e.g., major basic protein, eosinophil cationic protein, and leukotrienes) in the bronchial mucosa. A direct role for T lymphocytes in the development of airway hyperresponsiveness (i.e., independent of eosinophil recruitment) may also be important. Garssen and colleagues have shown that airway hyperresponsiveness can occur in a model of delayed-type hypersensitivity in the lung (30), and De Sanctis and coworkers have demonstrated that airway hyperresponsiveness can be transferred by T cell selective bone marrow transplantation in naive mice (25). The findings in the current report support the concept that airway hyperresponsiveness can occur independently of antigen-specific antibody responses and the mast cell dependent pathway. The recruitment of cells to sites of inflammation is a complex process that involves cell adhesion, activation, chemoattraction, and ultimately transmigration of the endothelial barrier. Chemokines play an important role in both the activation and chemoattraction phases of cellular recruitment. Chemokine expression in rodent models of allergen-induced pulmonary inflammation has been well described (7, 31 37). In the current experiments, both eotaxin and MCP-3, two chemokines important for eosinophil recruitment in this model (34, 38, 39), were upregulated in both wild-type and B cell deficient mice after immunization and challenge with antigen. In previous experiments, we demonstrated that eotaxin expression was upregulated in a similar fashion following antigen challenge (7). Furthermore, we demonstrated that elimination of T lymphocytes with anti T cell antibodies significantly diminished the expression of eotaxin in challenged mice without altering the levels of antigen-specific antibody titers. These findings suggest that in the absence of T cells, antigen-specific antibodies alone are not sufficient to induce the expression of eotaxin. The current findings demonstrate that at least two chemokines with activity for eosinophils can be upregulated in the absence of antigenspecific antibody responses, and these findings are consistent with our previous results. The importance of this result lies in determining the events that occur following the introduction of antigen into the airway of a sensitized animal. Humbles and colleagues have examined the kinetics of eosinophil recruitment and eotaxin expression in the guinea pig and have suggested that following allergen challenge, the mast cells may play an important role in the early events leading to eosinophil recruitment in normal mice (37). Our findings suggest that chemokine upregulation and eosinophil recruitment can occur independently of antigen-specific antibodies and that the T cell may play a central role in the regulation of these molecules. The current concept that pulmonary eosinophilia and airway hyperresponsiveness may arise by two separate pathways (i.e., mast cell and T cell) may also apply for chemokine induction in vivo. Further studies examining the in vivo regulation of chemokine expression in this model are ongoing. The finding that mice can develop antigen-induced pulmonary inflammation and airway hyperresponsiveness in the absence of antigen-specific antibodies is of interest. Whereas T cells are recognized as playing an important role in regulating asthmatic inflammation in humans, antigen-specific IgE antibodies and mast cells are also recognized as important mediators of allergic respiratory disease. Our study suggests that these are complicated models and that multiple possible parallels to asthmatic airway responses exist. Acknowledgments: The authors thank Zhi Qing Wu and Aiping Jiao for technical assistance. This work was supported by National Institutes of Health Grants AI01245 (J.A.M.), CA69212 and AI40618 (A.D.L.), and HL (J.M.D. and G.T.D.S.); Allen & Hanbury s Respiratory Diseases Research Award (J.A.M.); a grant from the Association Suisse Contre la Tuberculosle et les Maladies Respiratoires (A.S.); and a grant from the American Lung Association (G.T.D.S). References 1. Nakajima, H., I. Iwamoto, S. Tomoe, R. Matsumura, H. Tomioka, K. Takatsu, and S. 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