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Allergen-specific Th1 cells fail to counterbalance Th2 cell–induced airway hyperreactivity but cause severe airway inflammation
Gesine Hansen,1 Gerald Berry,2 Rosemarie H. DeKruyff,1 and Dale T. Umetsu1
1Division of Immunology and Transplantation Biology, Department of Pediatrics, and2Department of Pathology, Stanford University, Stanford, California 94305-5208, USA
Address correspondence to: Rosemarie H. DeKruyff, Department of Pediatrics, Room S323, Stanford University, Stanford, California 94305-5208, USA. E-mail: RDK@stanford.edu
Find articles by Hansen, G. in: JCI | PubMed | Google Scholar
1Division of Immunology and Transplantation Biology, Department of Pediatrics, and2Department of Pathology, Stanford University, Stanford, California 94305-5208, USA
Address correspondence to: Rosemarie H. DeKruyff, Department of Pediatrics, Room S323, Stanford University, Stanford, California 94305-5208, USA. E-mail: RDK@stanford.edu
Find articles by Berry, G. in: JCI | PubMed | Google Scholar
1Division of Immunology and Transplantation Biology, Department of Pediatrics, and2Department of Pathology, Stanford University, Stanford, California 94305-5208, USA
Address correspondence to: Rosemarie H. DeKruyff, Department of Pediatrics, Room S323, Stanford University, Stanford, California 94305-5208, USA. E-mail: RDK@stanford.edu
Find articles by DeKruyff, R. in: JCI | PubMed | Google Scholar
1Division of Immunology and Transplantation Biology, Department of Pediatrics, and2Department of Pathology, Stanford University, Stanford, California 94305-5208, USA
Address correspondence to: Rosemarie H. DeKruyff, Department of Pediatrics, Room S323, Stanford University, Stanford, California 94305-5208, USA. E-mail: RDK@stanford.edu
Find articles by Umetsu, D. in: JCI | PubMed | Google Scholar
Published January 15, 1999 - More info
J Clin Invest. 1999;103(2):175–183. https://doi.org/10.1172/JCI5155.
© 1999 The American Society for Clinical Investigation
Received: September 8, 1998; Accepted: November 18, 1998
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Methods
Animals. BALB/cBy mice were obtained from The Jackson Laboratory (Bar Harbor, Maine, USA), and histocompatible C.B-17Icrscid/scid mice (17) were obtained from the Stanford Medical Center Division of Laboratory Animal Medicine (Stanford, California, USA). DO11.10 mice, which are transgenic for TCR recognizing OVA peptide 323–339 (pOVA323–339) and backcrossed to BALB/c (18), were kindly provided by D. Loh and were bred in our facilities. Animals were used between 6 and 10 weeks of age and were age and sex matched within each experiment. All animal protocols were approved by the Stanford University Committee on Animal Welfare.
Monoclonal antibodies and reagents. Anti–IFN-γ monoclonal antibody (MAB) R46A2 (HB170, ATCC) and anti–IL-4 MAB (11B11) (both generously provided by J. Ohara and B. Paul, National Institutes of Health, Bethesda, Maryland, USA) were prepared from serum-free culture supernatants by ammonium sulfate precipitation. Anti–IL-2 MAB S4B6, anti–IFN-γ antibody XMG1.2, and anti–IL-5 MABs TRFK-4 and TRFK-5 were obtained from T. Mosmann (University of Alberta, Edmonton, Alberta, Canada). Anti–IL-4 MABs BVD4-1D11 and BVD6-24G2 were obtained from M. Howard (DNAX Research Institute, Palo Alto, California, USA). Each of these antibodies was purified from ascites by ammonium sulfate precipitation and ion-exchange chromatography. Neutralizing anti–IL-12 MAB C17.8, a generous gift of G. Trinchieri (The Wistar Institute, Philadelphia, Pennsylvania, USA), was purified from ascites by affinity chromatography. Hybridoma cells producing anticlonotype antibody KJ1-26.1 were generously provided by P. Marrack (National Jewish Medical Center, Denver Colorado, USA)(19). M. Howard (Anergen Inc., Redwood City, California, USA) generously provided murine rIL-4, and the Genetics Institute (Boston, Massachusetts, USA) generously provided rIL-12.
Preparation of OVA-specific Th1 and Th2 cells. OVA-specific Th1, Th2, and Th0 lines were generated from spleen and lymph node cells from DO11.10 (OVA TCR Tg) mice. T cells bearing the DO11.10 TCR were purified from spleen by negative selection on anti-Ig–coated plates, followed by positive selection using the anticlonotype antibody KJ1-26.1 and MACS anti–mouse IgG2a plus IgG2b microbeads. T cells were cultured at 5 × 105 per well with 4 × 106 irradiated spleen cells as Antigen-presenting celts (APC) and OVA (ICN Biomedicals, Costa Mesa, California, USA) (50 μg/ml). To generate Th2 lines, rIL-4 (40 U/ml) and anti–IL-12 MAB (10 μg/ml) were added to the cultures. Th0 lines were generated with the addition of rIL-4 (20 U/ml) and rIFN-γ (5 ng/ml), but not anti–IL-12, to the cultures. Th1 lines were generated by culturing T cells purified from lymph nodes of the DO11.10 mice in the presence of anti–IL-4 MAB and rIL-12 (0.1 ng/ml). After 7 days, cells were washed, counted, and restimulated with APC and antigen under the same conditions used for the initial stimulation. After the T cell lines achieved a stable committed phenotype (by the third cycle of stimulation), the T cell lines were maintained by weekly stimulation with APC and OVA without addition of anti-cytokine antibodies. Th1 lines were maintained with periodic addition of rIL-2. The cytokine profiles of the Th1, Th0, and Th2 lines were confirmed, by direct examination of supernatants generated from these lines, using ELISA for IL-4, IL-5, and IFN-γ, assays all well- established in our laboratory.
Transfer of cells. Th cell lines were transferred into histocompatible SCID mice. To facilitate homing of the T cell lines to the lungs, SCID mice were given 50 μg OVA in 50 μl NaCl 0.9% intranasally 1 day before intravenous transfer of cells. To facilitate pulmonary aspiration, mice were lightly anesthetized intraperitoneally with 0.25 ml of ketamine (0.44 mg/ml)/xylazine (6.3 mg/ml) in normal saline. Seventy-five percent of the intranasally administered antigen can be subsequently detected in the lungs (20).
Cultured Th1 or Th2 cells were harvested and washed with PBS. The results of pilot experiments suggested that transfer of 2.5 × 106 Th cells into syngeneic SCID recipients produced results similar to those observed when 5 × 106 or 7.5 × 106 Th cells were transferred. Therefore, 2.5 × 106 cells were adoptively transferred intravenously in most experiments. Some mice received a mixture of Th1 and Th2 cells (2.5 × 106 cells each or 5 × 106 Th1 plus 2.5 × 106 Th2 cells). Control mice received either OVA intranasally but NaCl 0.9% instead of T cells intravenously, or they received T cells intravenously but NaCl 0.9% instead of OVA intranasally. One and 2 days after the adoptive transfer of cells, OVA was again administered intranasally. Airway hyperreactivity was determined 24 h after the last intranasal dose of antigen was administered. Bronchoalveolar lavage (BAL) and lung fixation were performed the following day.
Immunization protocol of normal BALB/c mice to induce airway hyperreactivity. Th1 cell lines were also transferred into OVA-immunized BALB/c mice. BALB/c mice were immunized with OVA intraperitoneally (50 μg) complexed with aluminum potassium sulfate (alum) on days 1 and 14, and intranasally (50 μg OVA in 50 μl of PBS) on days 14, 25, 26, and 27. Control mice received intraperitoneal injections of alum alone and intranasal PBS. Some mice received Th1 cells intravenously (2.5 × 106 cells per mouse) on days 14 and 25. Airway hyperreactivity to inhaled methacholine was measured 24 h after the last intranasal dose of OVA (day 28). BAL and lung fixation were performed the following day (day 29).
Measurement of airway responsiveness. Airway responsiveness was assessed by methacholine-induced airflow obstruction from conscious mice placed in a whole-body plethysmograph (model PLY 3211; Buxco Electronics Inc., Troy, New York, USA). Pulmonary airflow obstruction was measured by Penh (see FDR RID="E1">). Penh = enhanced pause (dimensionless), Te = expiratory time, RT = relaxation time, PEF = peak expiratory flow (ml/s), and PIF = peak inspiratory flow (ml/s) (21). Enhanced pause (Penh), minute volume, tidal volume, and breathing frequency were obtained from chamber pressure, measured with a transducer (model TRD5100; Buxco Electronics) connected to preamplifier modules (model MAX2270; Buxco Electronics), and analyzed by system XA software (model SFT 1810; Buxco Electronics). Measurements of methacholine responsiveness were obtained by exposing mice for 2 min to NaCl 0.9% (Portable Ultrasonic 5500D; DeVilbiss Health Care Inc., Sommerset, Pennsylvania, USA), followed by incremental doses (2.5–40 mg/ml) of aerosolized methacholine and monitoring Penh. Results were expressed for each methacholine concentration as the percentage of baseline Penh values after NaCl 0.9% exposure.
(Equation 1)1
Collection of BAL fluid. Animals were injected intraperitoneally with a lethal dose of phenobarbital (450 mg/kg). After tying off the left lung at the mainstem bronchus, the trachea was cannulated; the right lung was then lavaged with 0.4 ml of PBS three times and the fluid pooled. Cells in the lavage fluid were counted using a hemocytometer, and BAL cell differentials were determined on slide preparations stained with Hansel Stain (Lide Laboratories, Florissant, Montana, USA). At least 200 cells were differentiated by light microscopy based on conventional morphologic criteria.
Lung histology. After washing with PBS, one part of the left lung was fixed in 10% formalin and stained with hematoxylin and eosin. The other part of the lung was embedded in OCT compound (Tissue Tec; Miles Inc., Elkhart, Indiana, USA), snap-frozen in liquid nitrogen, and stored at –80°C until sectioning. Then, 6-μm sections were fixed in acetone and stained with the biotinylated anticlonotypic antibody KJ1-26.1 and Streptavidin-FITC (PharMingen, San Diego, California, USA). A biotinylated anti–human IL-10 antibody (JES-312G8-biotinylated) was used as control antibody.
Cytokine ELISA. ELISAs were performed as described previously (22). The antibody pairs used were as follows, listed by capture/biotinylated detection: IFN-γ, R4-6A2/XMG1.2; IL-4, 11B11/BVD6-24G2; IL-5, TRFK-5/TRFK-4. The standards were recombinant cytokine curves generated in 1:2 dilutions from 20 to 0.156 ng/ml for IFN-γ, 500 to 7.5 pg/ml for IL-4, and 5,000 to 40 pg/ml for IL-5.
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Results
Generation and characterization of OVA-specific Th1, Th0, and Th2 cells. Th1, Th0, and Th2 cell lines were generated from OVA-specific T cells purified from the lymph nodes or spleen of OVA-specific TCR DO11.10 transgenic mice. CD4+ T cells were stimulated by OVA in the presence of rIL-4 and anti–IL-12 MAB to generate Th2 cell lines, whereas Th0 lines were generated by addition of rIL-4 and IFN-γ, but not anti–IL-12 MAB, to the culture. Th1 lines were generated by culturing OVA-specific T cells in the presence of anti–IL-4 MAB and rIL-12. They were maintained by weekly stimulation with irradiated APC and OVA, and Th1 lines required periodic addition of rIL-2. Figure 1 shows the cytokine content of supernatants from each Th cell line, as analyzed by ELISA for IL-4, IL-5, and IFN-γ. The Th1 cell lines produced high levels of IFN-γ but no detectable IL-4. In contrast, the Th2 cell lines produced high levels of IL-4 but no detectable IFN-γ. The Th0 cell lines produced both IFN-γ and IL-4. The quantity of IL-4 produced by the Th0 lines was significant, although less than that of Th2 lines. The Th1, Th2, and Th0 lines were used after at least three cycles of stimulation, after the T cell lines achieved a stable committed phenotype (23).
Figure 1Cytokine profiles of OVA-specific Th cell lines used in these studies. Th1, Th2, and Th0 cell lines (106 cells/ml) were stimulated with ConA (1 μg/ml) for 18 h. Supernatants were collected and analyzed by ELISA for IFN-γ, IL-4, and IL-5. IFN, interferon; IL, interleukin; OVA, ovalbumin.
In vivo transfer and detection of OVA-specific T cells to the lung. To determine the effects of Th cell lines on the development of airway hyperreactivity and airway inflammation, we adoptively transferred each cell line into histocompatible SCID mice. In these experiments, we chose SCID mice to eliminate the contribution of endogenous lymphocytes, which might inhibit the function of the transferred Th cells, and which could also contribute to airway hyperreactivity, as the recipients were exposed to OVA administered intranasally. To enhance the localization of the transferred T cell lines into the lungs of mice, we administered OVA intranasally one day before adoptive transfer of the T cell lines. One and two days later, OVA was again administered intranasally. Control mice received NaCl 0.9% instead of OVA intranasally. Four days after cell transfer, the lungs were stained with anti-TCR clonotypic antibody KJ1-26.1. Figure 2 shows that lungs from mice that received Th1 or Th2 cells and intranasal OVA stained brightly with the anti-TCR antibody, indicating that the OVA-specific T cell lines migrated to the lungs of these mice. In contrast, lungs from mice that received Th1 or Th2 cells, but no intranasal OVA, had no staining (data not shown), indicating that migration of the OVA-specific T cell lines was antigen dependent.
Figure 2OVA-TCR transgenic Th cells migrate to the lungs of recipient mice. Four days after adoptive transfer of OVA-specific Th cell lines (2.5 × 106 cells per mouse), mice were sacrificed and lung tissue was embedded in OCT compound. Frozen sections were obtained and stained with biotinylated anticlonotype antibody, KJ1-26.1 or biotinylated control antibody JES-312G8 and Streptavidin-FITC. (a) Lung tissue from recipient of Th1 cells. Left: Lung section stained with KJ1-26.1-biotin plus Streptavidin-FITC; Right: Lung section stained with biotinylated control antibody plus Streptavidin-FITC. (b) Lung tissue from recipient of Th2 cells. Left: Lung section stained with KJ1-26.1-biotin plus Streptavidin-FITC; Right: Lung section stained with biotinylated control antibody plus Streptavidin-FITC. TCR, T-cell receptor.
Airway histology in recipients of Th1, Th2, and Th0 cell lines. Mice were sacrificed four days after T-cell transfer. Lungs were removed and fixed, and lung sections were stained with hematoxylin and eosin to reveal lung histology. SCID mice that received OVA but no T cells had normal lung histology (Fig. 3a). Adoptive transfer of OVA-specific Th2 cells induced significant bronchiolar mucus production and induced peribronchiolar and perivascular infiltrates, consisting of lymphocytes, eosinophils, and some neutrophils (Fig. 3b). These results were similar to those reported in previous studies with Th2 cells (24). Transfer of OVA-specific Th1 cells also resulted in significant airway inflammation, with peribronchiolar and perivascular infiltrates (Fig. 3c). Th1 cells induced much less mucus production than did Th2 cells, and far more lymphocytes were present in the lungs when Th1 cells rather than Th2 cells were transferred. The inflammatory reaction with Th1 cells resembled that of moderate to severe lung allograft rejection, with dense perivascular and interstitial accumulations of small lymphocytes and immunoblasts with neutrophils and occasional eosinophils (25, 26). The inflammatory response with Th1 cells depended on the presence of OVA, as mice that received Th1 cells, but not OVA, had minimal lung disease (Fig. 3d). Furthermore, this inflammatory response with Th1 cells plus intranasal OVA was confined only to the lungs and was not observed in any other organ system.
Figure 3Histologic examination of lungs from SCID mice receiving Th1, Th2, and Th0 cells. (a) Lung tissue from control SCID mouse that received intranasal OVA but no Th cells. H&E, ×250. Inset: High-power magnification of normal bronchiolar epithelium. H&E, ×400. (b) Lung tissue from SCID mouse that received Th2 cells and intranasal OVA. Peribronchiolar mononuclear cell infiltrates are noted. The airway lumen is filled and expanded by thick mucus. H&E, ×250. Inset: High-power magnification of the airway epithelium showing tall columnar cells exhibiting abundant cytoplasmic mucin and a collarette of inflammatory cells. H&E, ×400. (c) Lung tissue from SCID mouse that received Th1 cells and intranasal OVA. Dense peribronchiolar inflammatory infiltrates are seen. The airway lumen does not contain mucus plugs. H&E, ×250. Inset: Lymphocytes are penetrating the airway epithelium and surrounding tissue spaces. H&E, ×400. (d) Lung tissue from control SCID mouse that received Th1 cells but not intranasal OVA. The bronchiole is normal with rare mononuclear cells in the peribronchiolar tissue; H&E ×250. Inset: The airway epithelium is normal. H&E, ×400. (e) Lung tissue from SCID mouse that received Th0 cells and intranasal OVA. Peribronchiolar infiltrates are noted, and the lumen is filled with mucus; scattered inflammatory cells are noted. H&E, ×250. Inset: The airway epithelium resembles that of mice that received OVA-specific Th2 cells, with the presence of tall columnar cells with abundant cytoplasmic mucin (b). (f) Lung tissue from SCID mouse that received both Th1 and Th2 cells and intranasal OVA. Significant airway inflammation is noted, without airway mucus. H&E, ×250. Inset: The epithelium displays reactive-appearing columnar cells, with inflammatory cells at the bases. H&E, ×400. H&E, hematoxylin and eosin; SCID, severe combined immunodeficiency.
Adoptive transfer of Th0 cell lines produced results similar to that observed with Th2 cell lines and induced significant airway inflammation (Fig. 3e). The production of IFN-γ by Th0 cells did not limit the inflammatory response, as large numbers of lymphocytes, neutrophils, and some eosinophils were present in the perivascular and peribronchiolar areas of Th0 cell recipients. Transfer of both Th1 and Th2 cells together did not result in reduction of airway inflammation, although the amount of bronchiolar mucus was reduced (Fig. 3f). This indicated that OVA-specific Th1 cells were not effective in counterbalancing Th2-induced airway inflammation.
Th2 cell–induced eosinophilia is significantly reduced by Th1 cells. The histopathologic analysis was extended by examination of the cell types and numbers in BAL fluid, which was harvested four days after adoptive T-cell transfer into SCID mice. The total number of cells recovered in the BAL for Th1-, Th2-, or Th1 plus Th2–treated mice was 4.2, 3.8, and 3.8 × 104/ml, respectively (Fig. 4). Macrophages were the predominant cell type in all groups of mice. Transfer of Th2 cells, however, significantly increased the proportion of eosinophils to 29.4% of total BAL cells (1.13 × 104 cells/ml), compared with 0.94% of total BAL cells (0.04 × 104 cells/ml) with Th1 cells. Transfer of a mixture of Th1 and Th2 cells in a ratio of 1:1 significantly decreased the number of eosinophils seen after transfer of Th2 cells from 29% to 12% of total BAL cells (0.47 × 104 cells/ml), suggesting that Th1 cells were able to reduce Th2 cell–induced airway eosinophilia. All other cell types together did not reach more than 2% in any group, and the proportions of these cell types did not differ significantly between the groups.
Figure 4OVA-specific Th1 cells significantly reduce the number of eosinophils induced by OVA-specific Th2 cells in BAL fluid of OVA-treated SCID mice. Four days after transfer of Th1 (2.5 × 106 cells per mouse), Th2 (2.5 × 106 cells per mouse), or Th1 plus Th2 (2.5 × 106 Th1 + 2.5 × 106 Th2 cells per mouse) cells in OVA-treated SCID mice, BAL was performed with three aliquots of 0.4 ml PBS per mouse (n = 6 for each group). The relative number of different types of leukocytes (lung cell differentials) was determined from Hansel Stain slide preparations of BAL fluid. The data are expressed as mean ± SEM of the percentage of each cell type derived from differentials based on 200 cells. Results are given as cells per milliliter in BAL fluid. BAL, bronchoalveolar lavage; Eos, eosinophils; Lym, lymphocytes; Mo, macrophages; Neu, neutrophils.
Th1 cells do not induce airway hyperreactivity. Three days after intravenous transfer of different T cell lines, we examined the recipients of Th1, Th2, and Th0 lines for airway hyperreactivity by challenging OVA-treated SCID mice with increasing concentrations of methacholine in a whole-body plethysmograph. Figure 5 shows that, as expected, SCID mice that had received Th2 lines developed significant airway hyperreactivity. Control SCID mice that received NaCl 0.9% rather than T cells intravenously and were challenged with intranasal OVA, or SCID mice that received Th2 cells intravenously but not intranasal OVA, did not develop airway hyperreactivity, indicating that this response depended on the presence of Th2 cells and on the presence of airway antigen. Recipients of Th0 cell lines developed significant airway hyperreactivity that was not statistically different from that induced with Th2 cells, suggesting that the production of IFN-γ by the T cells was not effective in reducing airway hyperreactivity. Transfer of Th1 lines, although inducing significant airway inflammation, did not induce airway hyperreactivity to methacholine. This indicated that airway hyperreactivity depends on factors produced by Th2-polarized cells and cannot be induced with inflammation-causing Th1-polarized cells.
Figure 5Th2 and Th0 cells, but not Th1 cells, increase airway hyperreactivity. SCID mice received Th1, Th2, or Th0 cells (2.5 × 106 cells per mouse) intravenously plus intranasal OVA. Control mice received either OVA only or cells only. Three days after adoptive cell transfer, airway hyperreactivity in response to increasing concentrations of inhaled methacholine was measured in a whole-body plethysmograph. Data are expressed as percent above baseline (mean ± SEM); n ≥ 7 for each data point. Cell transfer without intranasal administration of OVA had no effect on airway hyperreactivity (data not shown).
Th1 cells do not counterbalance airway hyperreactivity induced by Th2 cells. We next directly examined the capacity of Th1 cells to counterbalance airway hyperreactivity induced by Th2 cells by transferring a mixture of Th1 cells and Th2 cells into SCID mice. Figure 6 shows that although recipients of Th2 cells (2.5 × 106 cells per mouse) expressed significant airway hyperreactivity, recipients of Th1 cells plus Th2 cells in a ratio of 1:1 (2.5 × 106 cells each, Th1 + Th2 [1:1]) expressed equally strong airway hyperreactivity on methacholine challenge. Even on transfer of twice as many Th1 as Th2 cells (5 × 106 Th1 cells + 2.5 × 106 Th2 cells, Th1 + Th2 [2:1]), Th1 cells did not limit Th2 cell–induced airway hyperreactivity. These results indicate that antigen-specific Th1 cells do not inhibit airway hyperreactivity induced by Th2 cells even when given in a 2:1 excess.
Figure 6Th1 cells do not counterbalance airway hyperreactivity induced by Th2 cells. SCID mice received Th1 (2.5 × 106 cells per mouse) or Th2 (2.5 × 106 cells per mouse) cells intravenously plus intranasal OVA. Other SCID mice received a mixture of Th1 and Th2 cells in a ratio of 1:1 (2.5 × 106 cells each) or 2:1 (5 × 106 Th1 cells plus 2.5 × 106 Th2 cells) (n ≥ 5 for each data point). Airway hyperreactivity in response to inhaled methacholine was measured in a whole-body plethysmograph. Results are demonstrated as percent above baseline (mean ± SEM).
Th1 cells do not reduce airway hyperreactivity in OVA-immunized BALB/c mice. To determine whether Th1 cells could inhibit the development of Th2 cells in normal mice rather than reversing the function of established Th2 effector cells, we examined the capacity of adoptively transferred Th1 cell lines to prevent the development of allergen-induced airway hyperreactivity in normal BALB/c mice. Figure 7 shows that immunization of control BALB/c mice with OVA intraperitoneally and intranasally resulted in the development of significant airway hyperreactivity. Adoptive transfer of Th1 cells during the four-week sensitization phase on days 14 and 25 did not reduce this airway hyperreactivity, although Th1 cells migrated to the lungs as proved by fluorescence staining of lung sections with anti-TCR clonotypic antibody KJ1-26.1 one day after airway measurement (data not shown). These results indicated that Th1 cells were not able to inhibit the development of a Th2 response in sensitized immunocompetent BALB/c mice.
Figure 7OVA-specific Th1 cells do not reduce airway hyperreactivity in OVA-immunized BALB/c mice. BALB/c mice were immunized with OVA (50 μg) in alum intraperitoneally on days 0 and 14, and intranasally (50 μg OVA in 50 μl PBS) on days 14, 25, 26, and 27 (OVA; n = 6). A second group of mice also received OVA-specific Th1 cells intravenously on days 14 and 25 (2.5 × 106 cells per mouse at each time point) (OVA + Th1; n = 5). Control mice received alum intraperitoneally and PBS intranasally (no antigen; n = 6). Airway hyperreactivity to methacholine was determined as in Figs. 5 and 6. Results are expressed as mean ± SEM. Transfer of Th1 cells without administration of antigen did not have any effect on airway hyperreactivity (data not shown).
Airway eosinophilia in OVA-immunized BALB/c mice is significantly reduced by transfer of Th1 cells. Although Th1 cells were unable to reduce airway hyperreactivity in OVA-immunized mice, Th1 cells did significantly reduce airway eosinophilia (Fig. 8), indicating that the Th1 cells functioned in vivo. Th1 cells transferred during the sensitization phase reduced the number of BAL eosinophils by more than fivefold (from 13.5 × 105 to 2.3 × 105 eosinophils/ml), although the total number of cells in BAL fluid from OVA-sensitized BALB/c mice was not significantly reduced (18.1 × 105 cells/ml in BAL from control mice vs. 15.9 × 105 cells/ml in BAL from Th1-cell recipients). Lymphocytes and neutrophils were relatively rare in BAL fluid and did not differ significantly between the groups. However, histologic analysis revealed that the inflammatory response was not reduced by transfer of Th1 cells. Hematoxylin and eosin–stained lung sections of BALB/c mice after OVA immunization and Th1 cell transfer showed dense perivascular and peribronchiolar infiltrates containing numerous lymphocytes and eosinophils (data not shown). This pattern was comparable to the histology of the OVA-immunized control group.
Figure 8OVA-specific Th1 cells significantly reduce the number of eosinophils in OVA-immunized BALB/c mice. BALB/c mice were immunized with OVA (50 μg) in alum intraperitoneally on days 0 and 14 and intranasally (50 μg OVA in 50 μl PBS) on days 14, 25, 26, and 27 (OVA;n = 6). A second group of mice was immunized with OVA and additionally received OVA-specific Th1 cells intravenously on days 14 and 25 (2.5 × 106 cells per mouse at each time point) (OVA + Th1; n = 5). Control mice received alum intraperitoneally and PBS intranasally (no antigen; n = 6). BAL was performed on day 29 with three aliquots of 0.4 ml PBS per mouse, and the relative number of different types of leukocytes (lung cell differentials) was determined. The data are expressed as mean ± SEM of the percentage of each cell type derived from differentials based on 200 cells.
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