Abstract
We investigated the effects of in vivo intraperitoneal treatment with the rat monoclonal antibody (mAb), YN1.7.4 (YN1) against intercellular adhesion molecule-1 (ICAM-1) on the ovalbumin (OA)-inhalation-induced infiltration of leukocytes into the airways of OA-sensitized mice. YN1 (100 to 400 μg) given over a period of 72 h dose-dependently reduced the influx of lymphocytes and eosinophils into the bronchial lumen by > 60% and ⩾ 70%, respectively, when compared with saline or purified rat IgG-treated controls. Alveolar macrophages (AM) in the bronchoalveolar lavage fluid (BALF) were also decreased by > 50%. Lung tissue inflammation as determined by histopathologic examination was reduced. The number of neutrophils in the blood of OA-sensitized mice 3 days after challenge was significantly increased by treatment with YN1. However, at 24 h and 72 h after OA-challenge, the numbers of eosinophils and mononuclear cells in the bone marrow were reduced by YN1 treatment. Additionally, at 72 h after OA-challenge, the numbers of bone-marrow neutrophils were depressed. BALF levels of interleukin-5 (IL-5) and of IgA were lower for YN1-treated mice than for controls. With increasing doses of YN1, the levels of anti-ICAM-1 mAb in the plasma were proportionally increased. To correlate these results with YN1 treatment, blood and BALF T cells and BALF eosinophils were examined with flow cytometry. Blood T cells from YN1-treated mice were unable to bind phycoerythrin (PE)-labeled anti-ICAM-1 mAb ex vivo. These results implied that ICAM-1 on these cells was bound (occupied) by YN1 administered in vivo. Dose-related decreases were observed in the percentage and mean channel fluorescence (MCF) values of ICAM-1+ BALF T cells and eosinophils. The percentages of CD11a+ or CD49d+ eosinophils were also suppressed. Our data suggest that ICAM-1 is an important molecule involved in the recruitment of leukocytes into the airways of sensitized mice after pulmonary challenge.
Current understanding of the pathogenesis of bronchial asthma is based on evidence implicating chronic inflammatory mechanisms initiated by cytokines released by the Th2 subset of CD4+ T lymphocytes (1-5). One direct consequence of the proinflammatory cytokines produced is upregulation of the expression of adhesion molecules on endothelial, epithelial, and interstitial tissue in asthmatic airways. Adhesion molecules facilitate the recruitment of eosinophils, which are thought to be the final effector cells mediating the damage and subsequent remodeling of the airway wall in bronchial asthma (6-10).
Clinical and experimental data from bronchial biopsies of human asthma patients favor this hypothesis, with the observation that expression of adhesion molecules, such as intercellular adhesion molecule-1 (ICAM-1), is markedly enhanced (11-14). There is also a correlation between the severity of inflammation and the detection of soluble ICAM-1 in the peripheral blood and bronchoalveolar lavage fluid (BALF) of asthmatic individuals (15-17). A significant role for ICAM-1 in eosinophil recruitment into the lungs and bronchial hyperreactivity in a primate model of asthma was demonstrated with an anti-ICAM-1 monoclonal antibody (mAb) (18, 19). We have also reported corroborating evidence for the importance of ICAM-1 in a Brown Norway (BN) rat model of ovalbumin (OA)- induced allergic pulmonary inflammation, in which the anti-rat ICAM-1 mAb 1A29 was effective in reducing the infiltration of T cells and eosinophils into the BALF and lung tissue (20). These findings are in direct contrast to those in a study of mice, in which the movement of eosinophils and lymphocytes into the tracheas of OA-immunized mice was shown to be predominantly independent of the interaction between ICAM-1 and lymphocyte function-associated antigen-1 (LFA-1) (21).
With the present study we provide evidence for an important role for ICAM-1 in the accumulation of eosinophils and lymphocytes in the bronchial lumen and lung tissue after antigen inhalation in OA-sensitized mice. We have previously shown that the majority of the T cells in the airways of OA-sensitized mice are CD11a+ and ICAM-1+, and have increased flow-cytometric mean channel fluorescence (MCF) values when compared with splenic T cells (22). The number of alveolar macrophages (AM) was unchanged after OA challenge, but treatment with the anti-ICAM-1 mAb YN1 dramatically reduced the number of these leukocytes in the BALF. In order to determine that the reduction of pulmonary inflammation with YN1 may be correlated with direct evidence of YN1 binding to ICAM-1+ cells, flow cytometry was used to assess the binding of YN1 to ICAM-1+ leukocytes in the circulation and BALF fluid. YN1 selectively inhibited the influx of eosinophils, lymphocytes, and AM that were dependent on ICAM-1 on endothelial cells for trafficking into the airways, and the phenotypic characteristics of BALF T cells and eosinophils were also altered.
Female C57BL/6 mice were obtained from Jackson Laboratories (Bar Harbor, ME) and were used at 10 to 14 wk of age. OA of Grade V (Sigma Chemical Co., St. Louis, MO) was adsorbed to aluminum hydroxide gel (200 μg OA/180 mg Al(OH)3 in 4 ml saline) at 4°C overnight. Mice were immunized with 200 μl of the mixture given intraperitoneally. Vehicle-sensitized control mice were injected with an equal volume of a suspension containing no OA. Fourteen days later, mice were exposed for 10 min to an aerosol of OA generated from a 1.5% solution of the antigen dissolved in saline. Vehicle-sensitized and OA-challenged mice are designated as V-OA, and OA-sensitized and OA-challenged mice are indicated as OA-OA throughout this report.
All procedures in this study were in compliance with the Animal Welfare Act Regulations (9CFR Parts 1, 2, and 3) and with the Guide for the Care and Use of Laboratory Animals (DHEW Publication [NIH] 85-23, 1985).
The rat hybridoma YN1.7.4 (CRL1878, IgG2a) was obtained from American Type Culture Collection (ATCC; Rockville, MD). YN1 mAb was purified at Pharmacia and Upjohn, Inc. Vehicle- and OA-sensitized mice were injected intraperitoneally (100 μl/injection) with saline, purified rat IgG (Zymed, South San Francisco, CA), or YN1 mAb (in saline) at 12 h and 1 h before, and at 6 h, 24 h, and 48 h after aerosol challenge with OA. Mice in experiments that were terminated at 24 h or 72 h after challenge received a total of three or five intraperitoneal injections of YN1 or rat IgG, respectively.
Seventy-two hours after OA challenge, mice were anesthetized with 1.5 g/kg of urethane (Sigma) given intraperitoneally, and the trachea was cannulated. Lavage was performed by instilling 0.5 ml cold phosphate-buffered saline (PBS) into the lungs, which were gently massaged. The fluid was withdrawn and added to Ca2+- and Mg2+-free Hanks' balanced salt solution (HBSS) (Gibco BRL, Grand Island, NY) containing 5% fetal bovine serum (FBS) (HyClone, Logan, UT), 10 mM 4-(2-hydroxyethyl)-1-piperazine-N′-2-ethanesulfonic acid (Hepes) (Gibco), and antibiotics (penicillin–streptomycin, Gibco) (HBSS–FBS). Lavage was repeated with a second 0.5 ml of PBS and the collected fluid was pooled with the first BALF sample. The samples were centrifuged at 300 × g for 10 min, the BALFs were removed and stored at −20°C, and the cell pellets were resuspended in fresh HBSS–FBS. Total cell counts were determined for each sample with a Coulter counter Model ZM (Coulter Electronics, Hialeah, FL). Differential counts were made on cytospin preparations using a Shandon cytocentrifuge (Shandon Southern Instruments, Sewickley, PA). Cytospin slides were fixed and stained with Diff Quik (American Scientific Products, McGaw Park, IL). Differential counts were based on 200 cells, using standard morphologic criteria to identify the cells as neutrophils, eosinophils, lymphocytes, or monocytes/AM. The remaining BALF cells were pooled for immunofluorescence staining.
Mice were anesthetized, their tracheas were cannulated, and their lungs were lavaged 72 h after OA challenge. The lungs were removed and fixed by inflating with 10% phosphate-buffered formalin via the tracheal cannula to a pressure of 15 cm H2O. Specimens of trachea and the left lung were embedded in paraffin, sectioned at 6 μm, and stained with hematoxylin, phloxine, and eosin. The inflammatory reaction in the lungs was graded by a pathologist without knowledge of the group under study. Perivascular inflammatory-cell infiltrates and alveolar inflammation were scored separately on a scale of 0 to 5 (0 = none, 1 = minimal, 2 = mild, 3 = moderate, 4 = marked, 5 = severe). The percentage of eosinophils in the perivascular inflammatory-cell cuffs of each section was estimated and assigned a score ranging from 0 to 5 (0 = < 10%, 1 = 10 to 24%, 2 = 25 to 39%, 3 = 40 to 54%, 4 = 55 to 69%, 5 = 70%). This was not done for foci of alveolar inflammation, since they were infrequent. The total lung inflammation score was calculated by multiplying the perivascular inflammation score by the percent eosinophil score and adding the alveolitis score [total lung inflammation = (perivascular inflammation × % eosinophil score) + alveolitis].
Left femurs were removed and the leukocytes were flushed out with cold Dulbecco's modified Eagle's medium (DMEM) containing 10 mM Hepes, penicillin, and streptomycin. The leukocytes were washed by centrifugation through medium, and red blood cells were lysed with Tris-buffered ammonium chloride (0.02 M Tris, 0.14 M NH4Cl, pH 7.2). The cells were washed and resuspended in RPMI 1640 with 10% FBS. Cytospin slides were made and stained with Diff Quik. Differential counts were based on 200 bone marrow leukocytes, with the cells being classified as eosinophils, neutrophils or mononuclear cells.
Blood leukocytes were isolated by centrifugation of whole blood over separation medium. Briefly, 0.2-ml aliquots of blood from 10 mice were pooled, diluted to 5 ml with PBS, carefully layered over 5 ml of lymphocyte separation medium (Organon Teknika, Durham, NC), and centrifuged for 20 min at 500 × g. Mononuclear cells were harvested from the interface, and contaminating red blood cells were lysed with Tris-buffered ammonium chloride (0.02 M Tris, 0.14 M NH4Cl, pH 7.2). The following mAbs, either biotinylated or phycoerythrin (PE-) or fluorescein isothiocyanate (FITC-)conjugated, were purchased from PharMingen (San Diego, CA): 53-2.1 (Thy-1), 145-2C11 (CD3), 2D7 (CD11a), M1/70 (CD11b), R1-2 (CD49d), 3E2 (CD54), and MARK1 (anti-rat kappa light chain). Approximately 5 × 105 pooled BALF or blood cells/well (96-well round-bottom plates) were stained with mAbs diluted in phenol red-free HBSS containing 2% FBS, 10 mM Hepes, and 0.02% Na azide. Nonspecific background staining was reduced by pretreatment of samples with anti-CD32 (FcγIIR) mAb. Cells were then incubated with conjugated mAb for 45 min on ice and washed three times. Biotinylated mAb was developed with Texas Red–streptavidin (Molecular Probes, Eugene, OR) through incubation for an additional 30 min. Controls were generated by staining cells in the same manner with conjugated irrelevant myeloma Ig. After three washes, cells were fixed in PBS containing 1% paraformaldehyde and 10 mM Hepes. Samples were analyzed with a Coulter EPICS 753 flow cytometer equipped with a 5-W argon laser for excitation of FITC and PE, and a rhodamine-6G dye laser for excitation of Texas Red. Fluorescence signals were logarithmically amplified over a three-decade range in 256 channels for single-parameter data and in 64 × 64 channels for two-parameter data. Live/dead, eosinophil and lymphocyte gates were set on the basis of forward angle and 90° light-scatter signals, verified through propidium iodide exclusion. T cells were identified by costaining with Thy-1 and CD3 mAb. Data were acquired in list mode on ⩾ 15,000 gated events and analyzed with the Elite software package (Coulter Corporation). MCF values from log scales were converted to linear mean channel values for direct comparison of different intensities (23).
Lungs were lavaged as described 3 days after OA challenge, and the samples were assayed for the level of murine interleukin-5 (IL-5) through the use of mAb pairs from PharMingen.
The concentration of IgA in BALF fluid was measured with an enzyme-linked immunosorbent assay (ELISA). Half-area 96-well enzyme immunoassay (EIA) plates were coated overnight at 4°C with antimouse IgA (The Binding Site Ltd, Birmingham, UK) in 0.02 M carbonated buffer (pH 9.6), in a volume of 50 μl/well. Unbound protein was removed by washing three times with Dulbecco's phosphate-buffered saline (DPBS) containing 0.05% Tween-20 (DPBS/TW). The wells were blocked for 1 h at room temperature (RT) with 100 μl/well DPBS/TW containing 1% bovine serum albumin (BSA) (DPBS/TW/BSA). After three washes with DPBS/TW, 50 μl/well of BALF was added. A standard curve was constructed from mouse IgA kappa (Sigma) diluted in DPBS/TW/BSA (serial 3-fold dilutions, starting at 100 μg/ml) and added for 2 h to 3 h at RT. The wells were washed three times and incubated for 1 h at RT with 50 μl/well alkaline phosphatase-labeled goat antimouse IgA (Southern Biotechnology Associates, Inc., Birmingham, AL) diluted in DPBS/TW/BSA. The appropriate dilution of this antibody was predetermined to have little cross-reactivity with other serum isotypes. Unbound antibody was removed through three washes, and color was developed with the addition of 50 μl/well of 1 mg paranitrophenylphosphate (Substrate No. 104; Sigma) per milliliter of Tris–sodium barbital buffer (Gelman, Ann Arbor, MI). After incubation for 30 to 60 min at RT, absorbance (405 nm) was read with an automated plate reader (Molecular Devices, Inc., Menlo Park, CA). The concentration of IgA in each BALF sample was calculated from the standard curve, using the Immunofit curve fitting program (Beckman Instruments, Palo Alto, CA).
Data were analyzed through one-way analysis of variance (ANOVA). When this overall test of treatment was found to be significant (P < 0.05), two-sided pairwise comparisons were made with the least-significant-difference (LSD) method (24). Data that were significantly (P < 0.05) different are denoted by a hatchmark (#). The ANOVA with LSD comparisons was also performed on ranked-transformed data. Significant differences on ranked data for P < 0.05 are indicated by an asterisk (*).
Incremental increases in YN1 dosed at 100, 200, or 400 μg/ injection produced mean plasma rat IgG concentrations of 68.9 ± 35.3, 327.8 ± 239, and 548.5 ± 144 μg/ml (mean ± SD), respectively, at 72 h after OA inhalation. Similarly, administration of rat IgG at the same doses yielded higher mean plasma concentrations of 261.9 ± 81.7, 605 ± 154, and 666.8 ± 176 μg/ml, respectively. With both rat IgG and YN1, the mean plasma concentrations observed with the higher two doses of antibodies were significantly (P < 0.05) greater than that obtained with 100 μg/dose.
Inhalation of OA by sensitized mice increased the total number of leukocytes in the BALF by almost 3-fold as compared with that in V-OA mice (Figure 1A). The data showed that treatment with YN1 at 100 and 200 μg/dose significantly reduced the total number of leukocytes in the BALF (Figure 1A). Administration of purified rat IgG (at 100 or 200 μg/dose) did not affect the total number of cells in the BALF as compared with that in mice treated with saline.
Fig. 1. Effect of anti-ICAM-1 mAb YN1 on accumulation of eosinophils, lymphocytes, and neutrophils in the bronchial lumen of OA-sensitized and -challenged C57BL/6 mice. Immunized mice were injected five times intraperitoneally with saline, purified rat IgG, or YN1 (100 or 200 μg/mouse/injection). The lungs were lavaged 72 h after OA challenge, and the mean total number of leukocytes ± SEM in the BALF for groups of 10 to 20 mice was calculated (A). (B) Numbers of neutrophils, eosinophils, and lymphocytes in the BALF. Significant differences (P < 0.05) in means for OA-OA groups treated with purified rat IgG versus groups tested with YN1 are indicated by # and * for ANOVA on untransformed and rank-transformed data, respectively.
[More] [Minimize]Figure 1B shows that the influx of eosinophils into the bronchial lumen was decreased by 63% and 78% with 100 and 200 μl of YN1, respectively. Similarly, the lymphocyte population was reduced by 60 to 70% with both doses of YN1. The number of neutrophils, which comprise a minor subset of the leukocytes in BALF, was also diminished after treatment with 200 μg/injection of YN1.
The number of AM in BALF from V-OA and OA-OA mice treated with saline was similar. Furthermore, intraperitoneal injections with purified rat IgG at 100 or 200 μg/dose did not have any apparent effect on this cell population (Figure 2). Upon treatment with YN1 at either 100 or 200 μg/injection, the numbers of AM in the BALF of OA-OA mice were depressed (P < 0.05) by 60% and 54%, respectively, when compared with those of similar groups of mice that were injected with purified rat IgG.
Fig. 2. Effect of anti-ICAM-1 mAb YN1 on AM in BALF of OA-sensitized and -challenged C57BL/6 mice. Immunized mice were injected five times intraperitoneally with saline, purified rat IgG, or YN1 (100 or 200 μg/mouse/injection), and 72 h after OA challenge, AM in BALF were enumerated. The data are the mean number of macrophages ± SEM for groups of 10 mice. Significant differences (P < 0.05) in the means for OA-OA groups treated with purified rat IgG or YN1 are indicated by # and * for ANOVA on untransformed and ranked data, respectively.
[More] [Minimize]The lungs of the majority of the mice in the V-OA group treated with saline lacked an appreciable inflammatory response (Figure 3A). Some of the mice in this group, however, had cuffs of inflammatory cells around scattered pulmonary blood vessels. The inflammatory infiltrate was composed of a mixed population of mononuclear cells including lymphocytes, macrophages, and plasma cells. Eosinophils were infrequently noted. The total lung inflammation score for this group was 0.2 ± 0.2 (mean ± SEM). Lungs from OA-OA mice injected with either saline or purified rat IgG also showed inflammatory-cell cuffs around multiple blood vessels. In these mice, the presence of the perivascular inflammatory infiltrate was more consistent and more intense than that observed in the V-OA groups (Figure 3B). Furthermore, eosinophils were frequently a prominent component of the perivascular cuffs. This was especially true of inflammatory-cell cuffs located around small arteries. The inflammatory-cell cuffs around small veins tended to be dominated by mononuclear cells. Scattered foci of alveolar inflammation sometimes occurred. The lung inflammation scores for the OA-OA group given either saline or purified rat IgG were 4.9 ± 1.22 and 4.8 ± 1.32, respectively. In contrast, lungs from OA-OA mice injected with YN1 (200 μg/dose) exhibited decreases in lung inflammation (Figure 3C). In these mice, inflammatory-cell infiltrates were found around scattered pulmonary blood vessels; however, eosinophils were only occasionally a prominent part of this process. More often, the perivascular cuffs were composed almost entirely of mononuclear cells. The lung inflammation score for the YN1-treated group was 2.8 ± 1.2.
Fig. 3. Histologic assessment of lung tissue of mice treated with YN1, purified rat IgG, or saline. The lung tissue was collected from OA-immunized mice 72 h after OA challenge. (A) Lung from a V-OA mouse that was injected with saline. A small blood vessel (center of photograph) is surrounded by inflammatory cells. Note that these are mononuclear cells. Eosinophils are not present. Bar = 25 μm. (B) Lung from an OA-OA mouse treated five times with purified rat IgG (200 μg/ dose). Inflammatory cells are present around a small blood vessel (center of photograph). Note that many of these cells are eosinophils (arrows). Bar = 25 μm. (C) Lung from an OA-OA mouse injected five times with YN1 (200 μg/dose). A small blood vessel (center of photograph) is partially surrounded by mononuclear cells, and was similar to that observed in V-OA mice. Eosinophils are not present. Bar = 25 μm.
At 24 h and 72 h after OA challenge of sensitized mice, the composition of blood leukocytes in mice treated with 200 μg/dose/mouse of rat IgG or YN1 was determined. Table 1 shows that no detectable changes in the leukocyte profile of YN1-treated mice were apparent when the cells were collected at 24 h after OA inhalation. A significant increase in the number of neutrophils in the blood of YN1-treated mice, however, was noted at 72 h.
| Number × 10−4/ml ± SEM | ||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Hours After Challenge | Treatment | Eosinophils | Neutrophils | Monocytes | Lymphocytes | Basophils | ||||||
| 24 | IgG | 8.3 ± 1.8 | 63.8 ± 8.2 | 16.8 ± 2.9 | 405 ± 38 | 2.4 ± 0.4 | ||||||
| YN1 | 6.0 ± 1.1 | 71.0 ± 5.2 | 13.5 ± 1.3 | 419 ± 50 | 2.6 ± 0.5 | |||||||
| 72 | IgG | 13.4 ± 1.9 | 56.9 ± 5.8 | 14.7 ± 1.3 | 371 ± 51 | 3.1 ± 0.9 | ||||||
| YN1 | 13.5 ± 2.1 | 90.9 ± 8.2* | 17.4 ± 3.7 | 377 ± 39 | 2.1 ± 0.5 | |||||||
At 24 h and 72 h after inhalation of antigen by sensitized mice treated with rat IgG or YN1, the left femurs were removed and the bone-marrow leukocytes were enumerated (Table 2). The number of bone marrow mononuclear cells and eosinophils in YN1-treated mice was found to be significantly smaller than that of controls when examined at 24 h after challenge. At the later time point of 72 h after challenge, all three leukocyte subsets (mononuclear cells, eosinophils, and neutrophils) were diminished in mice given YN1 as compared with OA-OA mice treated with rat IgG.
| Hours After Challenge | Number × 10−6 ± SEM | |||||||
|---|---|---|---|---|---|---|---|---|
| Treatment | Eosinophils | Neutrophils | Monocytes | |||||
| 24 | IgG | 1.52 ± 0.11 | 4.36 ± 0.29 | 17.35 ± 0.76 | ||||
| YN1 | 1.12 ± 0.11* | 4.12 ± 0.63 | 13.61 ± 1.56* | |||||
| 72 | IgG | 1.41 ± 0.14 | 2.96 ± 0.35 | 7.47 ± 0.86 | ||||
| YN1 | 0.61 ± 0.06** | 1.71 ± 0.10** | 4.57 ± 0.18** | |||||
Blood from OA-OA mice treated with saline, rat IgG, or YN1 was collected 72 h after aerosol challenge, and mononuclear cells were isolated. In all groups, 30 to 40% of the cells were T cells, as identified by Thy-1. Thy-1+CD3+-ICAM-1+ T cells obtained from the circulation of OA-OA mice treated with saline or purified rat IgG at 100 and 200 μg/dose ranged from 26 to 35%, with MCF values of ∼ 40 (Table 3). After in vivo administration of YN1, ⩽ 4% of the Thy-1+CD3+ T cells in the blood were detectable with PE-labeled anti-ICAM-1 mAb, with concomitant decreases in the MCF values. The numbers of lymphocytes in the peripheral blood after YN1 treatment were no different than the numbers for mice given saline or rat IgG (data not shown). With a murine anti-rat kappa mAb (MARK1), binding of rat mAb was detected on ∼ 30% of blood T cells from mice treated with 200 μg/dose of YN1. Although the percentage of blood T cells from anti-ICAM-1 mAb-treated mice that were CD11a+ or CD49d+ remained constant as compared with those from mice given saline or purified rat IgG, the MCF values for CD11a staining were reduced by 30% with both doses of YN1.
| In Vivo Treatment (μg/dose/mouse, intraperitoneally 5×) | ||||||||||
|---|---|---|---|---|---|---|---|---|---|---|
| Rat IgG | Anti-ICAM-1 mAb (YN1) | |||||||||
| Marker | Saline | 100 | 200 | 100 | 200 | |||||
| CD54 | 25.6 | 35.0 | 34.0 | 3.8 | 2.3 | |||||
| (39.2) | (42.2) | (41.9) | (26.5) | (26.7) | ||||||
| CD11a | 69.8 | 73.0 | 73.6 | 68.3 | 65.3 | |||||
| (56.7) | (69.2) | (66.5) | (50.8) | (46.4) | ||||||
| CD49d | 23.3 | 20.9 | 27.3 | 24.4 | 26.4 | |||||
| (α4) | (38.3) | (37.8) | (39.8) | (37.7) | (35.1) | |||||
The BALF leukocytes obtained at 72 h after antigen challenge from immunized mice that were injected with purified rat IgG or YN1 were pooled and analyzed with flow cytometry (Table 4). There was an inverse relationship between the percentages and MCF values of Thy-1+CD3+ICAM-1+ T cells in the BALF and the doses of YN1 given to OA-OA mice in vivo. The percent detectable ICAM-1+ T cells fell to 23% and 16% from ∼ 50% with 100 and 200 μg/doses of YN1, respectively. The percentages of CD11a+ and CD49d+ T cells, respectively, in the BALF were unchanged, but the MCF values for CD11a and CD49d expression were diminished by 20%. BALF T cells did not bind the murine anti-rat mAb MARK1 (data not shown).
| In Vivo Treatment (μg/dose/mouse, intraperitoneally 5×) | ||||||||
|---|---|---|---|---|---|---|---|---|
| Rat IgG | Anti-ICAM-1 mAb (YN1) | |||||||
| Marker | 100 | 200 | 100 | 200 | ||||
| CD54 | 55.5 | 51.8 | 23.0 | 15.9 | ||||
| (37.7) | (42.6) | (29.4) | (29.7) | |||||
| CD11a | 68.7 | 48.5 | 73.1 | 63.1 | ||||
| (62.8) | (64.2) | (48.0) | (50.1) | |||||
| CD49d | 39.1 | 43.0 | 45.4 | 40.5 | ||||
| (α4) | (49.9) | (48.6) | (47.0) | (39.5) | ||||
The phenotypic characteristics of the BALF eosinophils from OA-OA mice after YN1 administration were also examined (Table 5). The percentages and intensities of staining of ICAM-1+ eosinophils in the BALF of mice treated with saline or either dose of purified rat IgG were not different (data not shown). However, with increasing concentrations of YN1 administered in vivo, a dose-dependent decrease in the percentage of ICAM-1+ eosinophils was observed. Furthermore, CD11a+ and CD49d+ eosinophils were reduced by 28% and 20%, respectively, at the 200 μg/dose of YN1, whereas the MCF values for CD11a, CD49d, and CD54 expression were enhanced. The percentage of CD11b+ BALF eosinophils was also decreased at the higher of the two YN1 doses.
| In Vivo Treatment (μg/dose/mouse, intraperitoneally 5×) | ||||||||
|---|---|---|---|---|---|---|---|---|
| Rat IgG | Anti-ICAM-1 mAb (YN1) | |||||||
| Marker | 100 | 200 | 100 | 200 | ||||
| CD54 | 65.4 | 67.0 | 51.3 | 33.4 | ||||
| (45.1) | (48.9) | (41.2) | (56.8) | |||||
| CD11a | 83.0 | 79.7 | 73.7 | 57.3 | ||||
| (147) | (145) | (134) | (229) | |||||
| CD11b (Mac-1) | 94.9 | 92.4 | 91.0 | 86.4 | ||||
| CD49d (α4) | 52.4 | 46.6 | 53.3 | 38.1 | ||||
| (46.9) | (52.0) | (54.0) | (81.6) | |||||
To assess whether administration of the anti-ICAM-1 mAb YN1 may have had an effect on cellular events preceding the accumulation of leukocytes in the airways after OA challenge, we determined the concentrations of IL-5 and IgA in BALF of control and YN1-treated mice. The lungs of sensitized mice treated with either control rat IgG or YN1 at 200 μg/dose/mouse were lavaged at 24 h after OA challenge. The level of IL-5 in BALF samples from OA-sensitized and -challenged mice was almost 8-fold greater than that of nonsensitized controls (Figure 4A). Treatment with YN1 significantly reduced the concentration of IL-5 in BALF.
Fig. 4. Effect of YN1 treatment on the level of BALF IL-5 and IgA in OA-sensitized and -challenged mice. Twenty four hours after OA inhalation, the lungs of sensitized mice treated with control rat IgG or YN1 at 200 μg/dose/mouse were lavaged. The BALF samples were analyzed for the concentration of (A) IL-5 and (B) IgA as described. The data were analyzed with ANOVA, and the mean ± SEM for groups of 10 mice are shown. Significant differences between V-OA mice injected with control rat IgG versus OA-OA mice treated with YN1, and between OA-OA mice treated with YN1 versus rat IgG, are indicated by * or # for rank or raw transformed data at P < 0.05.
[More] [Minimize]Similar observations were made with respect to the amount of IgA in BALF that was detectable with ELISA (Figure 4B). OA challenge of sensitized mice increased the concentration of IgA in BALF, and this increase was dampened by treatment with YN1.
The focus of our study was on assessing the involvement of ICAM-1 in leukocyte trafficking in a murine model of pulmonary inflammation that is dominated by the infiltration of LFA-1+ and ICAM-1+ eosinophils and lymphocytes into the airways. After in vivo administration of an anti-ICAM-1 mAb, YN1, we showed incremental increases in the level of circulating YN1 as the doses were raised from 100 to 400 μg/dose/mouse. The resulting concentrations of circulating YN1 have been shown to be sufficient to block the binding of leukocytes to the endothelium. As in the rat model of OA-induced pulmonary inflammation (20), neutrophilia in the peripheral blood was observed in our model. However, the increase in the number of circulating neutrophils was measurable only at 72 h, and not at 24 h after challenge. It is noteworthy that none of the other four leukocyte subsets (monocytes, lymphocytes, basophils, or eosinophils) was changed by YN1 treatment. Examination of bone-marrow leukocyte subsets from femurs revealed a decrease in the number of leukocytes at 24 h and 72 h after antigen challenge. It is possible that the decreases in the leukocytes in the bone marrow are be caused by the release of leukocytes into the circulation, and that this may have contributed to the increase in blood neutrophils.
When blood T cells from YN1-treated mice were examined with flow cytometry, however, there was a profound decrease in the percentage of Thy-1+ CD3+ T cells that were still able to bind ICAM-1 mAb ex vivo. This suggested that almost 90% of the ICAM-1+ blood T cells may have had YN1 bound to their surface. This was further supported by our observation that these blood T cells were detectable with a murine antirat IgG mAb when they were examined ex vivo. From these data it may be inferred that YN1 had also interacted with the majority of the ICAM-1 molecules on nonleukocytes, such as pulmonary endothelial cells, in vivo in the presence of high levels of detectable YN1 in the circulation.
Since we were interested in ICAM-1-dependent leukocyte recruitment in the airways, the reductions in BALF leukocyte numbers clearly demonstrated that ICAM-1 was an important molecule in the OA-elicited accumulation of eosinophils and lymphocytes. Our data with C57BL/6 mice are consistent with results in sensitized BN rats, in which an anti-rat ICAM-1 mAb, 1A29, also was found to inhibit OA-induced eosinophil- and lymphocyte-rich inflammation in the airways (20). Administration of YN1 to mice in our model did not produce leukopenia or prevent the release of bone marrow leukocytes into the circulation. Therefore, it is unlikely that the inhibition of OA-elicited BALF leukocyte accumulation by YN1 was simply a consequence of changes in the distribution of leukocytes in the systemic circulation. The histopathology of the lung tissue in our study also revealed an attenuation of eosinophilic and mononuclear-cell inflammation by YN1. These results are in direct contrast to those in a recent report by Nakajima and colleagues (21) in which ICAM-1 was not found to be important for the infiltration of eosinophils into the tracheas of OA-sensitized mice even when doses as high as 4 mg of YN1 were used. However, their data, and our present study, did show that the movement of T cells into the airways (trachea and bronchial lumen, respectively) was inhibited by YN1. We were able to achieve a slightly greater inhibition of eosinophils of almost 80%, whereas maximal blockage of T-cell recruitment was only ∼ 70% with 200 μg/dose of YN1.
The number of AM in the BALF of OA-sensitized mice was similar to that of normal unimmunized controls, and was not increased by antigen challenge. This “resident” population of AM was substantially decreased after treatment with YN1. Although we do not show data for the expression of LFA-1 or ICAM-1 on murine AM, enhanced expression of ICAM-1 has been reported for human AM recovered by BALF from asthmatic subjects (25). There is also evidence that migration of monocytes into inflammatory lesions in the skin or arthritic joints in the rat is perturbed by mAbs against leukocyte integrins LFA-1 and Mac-1, two ligands for ICAM-1 (26). Whether the inhibition of interactions between ICAM-1 and CD11a will have a deleterious effect on the number or activation of AM in human asthmatic subjects will require further study.
In order to show that the reduction of BALF eosinophils and T cells and of lung pathology associated with YN1 treatment was a direct consequence of the interaction of YN1 with ICAM-1+ cells, we examined the phenotypic characteristics of BALF T cells and eosinophils with flow cytometry. We have used the analysis of ICAM-1+ peripheral blood and BALF T cells as a tool to further our understanding of the potential changes associated with in vivo treatment with YN1. It was apparent that there was a dose-related decrease in our ability to detect Thy-1+- CD3+ICAM-1+ BALF T cells ex vivo, concomitant with a reduction of the MCF value. Detectable ICAM-1+ BALF T cells were reduced by 50% and 70% at 100 and 200 μg/ doses of YN1, respectively, as compared with those of mice treated with purified rat control IgG. In contrast to blood T cells, BALF T cells from YN1-treated mice did not bind the murine antirat IgG mAb, indicating that the BALF T cells that were able to enter the airways did not have YN1 bound to their surface. Concomitant with the progressive, dose-dependent loss of ICAM-1+ BALF T cells, the intensities of staining of CD11a and CD49d were also reduced with YN1 treatment. The data suggest that the phenotypic characteristics of the BALF T cells from YN1 treated mice were different from those of control animals. Since we have previously observed that the coexpression of ICAM-1, CD11a, and CD49d on BALF T cells recruited into the airways after antigen provocation was clustered on discrete subsets of BALF T cells (22), we speculate that these results may indicate a selective inhibition by YN1 of subpopulations of T cells that may express more than one of these adhesion molecules. It was also interesting to note that the expression of CD11a on blood T cells from YN1-treated mice was less intense than on blood T cells from control mice. It is unclear whether this indicates that ICAM-1/CD11a interactions are necessary as positive signals for augmenting the expression of CD11a on T cells.
We have presented data showing that murine eosinophils found in the lung also expressed CD54, CD49d, and CD11a. In parallel with its effects on T-cells, treatment with YN1 led to reductions in the percentage of detectable ICAM-1+ eosinophils in BALF. Interestingly, at the higher dose of 200 μg, more changes in the phenotypic profile of BALF eosinophils were detected with respect to CD11a and CD49d. In addition to decreased percentages of CD11a+ and CD49d+ BALF eosinophils with YN1 treatment, there were measurable increases in MCF values. This may imply that the eosinophils that were able to emigrate into the airways despite YN1 treatment were able to do so because of the expression of higher levels of these two (and potentially other) adhesion molecules as a compensatory mechanism (6, 27). It may be extrapolated that binding of YN1 to ICAM-1 on endothelial cells occurs in a manner similar to that seen with T cells and eosinophils. In the present study we confined our attention to ICAM-1, and did not address the potential role of ICAM-2 and ICAM-3, two other counterreceptors for LFA-1 and Mac-1 binding (28, 29), or that of adhesion-dependent cellular activation (30-32), and their effects on leukocyte trafficking in the lung.
It must be emphasized, however, that the resolution of antigen-induced pulmonary inflammation by YN1 is probably multifaceted, involving disruption of the accessory role of ICAM-1/LFA-1 in T-cell activation (33), as well as disruption of the binding of leukocytes to the activated pulmonary endothelium. We recently identified both IL-5 messenger ribonucleic acid (mRNA) and protein in lung tissue and BALF of OA-sensitized and -challenged mice (34). A number of reports have described the importance of this cytokine in murine models of pulmonary eosinophilia (35, 36). Therefore, the decrease in BALF of IL-5, a cytokine important in the recruitment of eosinophils, may suggest that ICAM-1 is important in initiating the release of IL-5 from T cells shortly after exposure to antigen. It is probably unlikely that the IL-5 in the BALF of sensitized mice 24 h after challenge is produced by eosinophils, since the release of IL-5 precedes the infiltration of eosinophils. A further indication that YN1 perturbs more than the adherence of leukocytes to the endothelium is by our finding that BALF IgA levels were lower than those of control mice after inhalation of OA. There is published evidence supporting a role for secretory IgA in the activation and degranulation of eosinophils in patients with allergies and asthma (37, 38), and receptors for IgA (FcαR) and their enhanced expression in individuals with allergies have been identified (38, 39). The precise nature by which YN1 intervenes in the synthesis of IgA by B cells remains to be resolved.
Our observation that OA-induced recruitment of eosinophils and lymphocytes into BALF is blocked by YN1, with a concomitant decrease in the number of AM and a resolution of lung pathology, leads us to conclude that ICAM-1 has an important role in generating an inflammatory response in a murine model of allergic, eosinophil-rich lung inflammation. Therapeutic intervention aimed at regulating the expression and function of ICAM-1 in the airway may prove efficacious for treating lung diseases such as asthma.
| 1. | Azzawi M., Bradley B., Jeffery P. K., Frew A. J., Wardlaw A. J., Knowles G., Assoufi B., Collins J. V., Durham S., Kay A. B.Identification of activated T lymphocytes and eosinophils in bronchial biopsies in stable atopic asthma. Am. Rev. Respir. Dis.142199014071413 |
| 2. | Robinson D., Hamid Q., Bentley A., Ying S. T., Kay A. B., Durham S. R.Activation of CD4+ T cells, increased TH2 type cytokine mRNA expression, and eosinophil recruitment in bronchoalveolar lavage after allergen inhalation challenge in patients with atopic asthma. J. Allergy Clin. Immunol.921993313324 |
| 3. | Del Prete G. F., De Carli M., D'Elios M. M., Maestrelli P., Ricci M., Fabbri L., Romagnani S.Allergen exposure induces the activation of allergen-specific Th2 cells in the airway mucosa of patients with allergic respiratory disorders. Eur. J. Immunol.23199314451449 |
| 4. | Romagnani S.Lymphokine production by human T cells in disease states. Annu. Rev. Immunol.121994227257 |
| 5. | Barnes P. J.Cytokines as mediators of chronic asthma. Am. J. Respir. Crit. Care Med.1501994S42S49 |
| 6. | Springer T. A.Traffic signals for lymphocyte recirculation and leukocyte emigration: the multistep paradigm. Cell761994301314 |
| 7. | Canonica G. W., Ciprandi G., Buscaglia S., Pesce G., Bagnasco M.Adhesion molecules of allergic inflammation: recent insights into their functional roles. Allergy491994135141 |
| 8. | Pilewski J. M., Albelda S. M.Cell adhesion molecules in asthma: homing activation, and airway remodeling. Am. J. Respir. Cell Mol. Biol.12199513 |
| 9. | Kroegel C., Virchow J.-C., Luttman W., Walker C., Warner J. A.Pulmonary immune cells in health and disease: the eosinophil leukocyte (Part I). Eur. Respir. J.71994519543 |
| 10. | Jones D. G.The eosinophil. J. Comp. Pathol.1081993317335 |
| 11. | Bentley A. M., Durham S. R., Robinson D. S., Menz G., Storz C., Cromwell O., Kay A. B., Wardlaw A. J.Expression of endothelial and leukocyte adhesion molecules intercellular adhesion molecule-1, E-selectin, and vascular cell adhesion molecule-1 in the bronchial mucosa in steady-state and allergen-induced asthma. J. Allergy Exp. Immunol.921993857868 |
| 12. | Manolitsas N. D., Trigg C. J., McAulay A. E., Wang J. H., Jordan S. E., D'Ardenne A. J., Davies R. J.The expression of intercellular adhesion molecule-1 and the β1-integrins in asthma. Eur. Respir. J.7199414391444 |
| 13. | Gosset P., Tillie-Leblond I., Janin A., Marquette C.-H., Copin M.-C., Wallaert B., Tonnel A.-B.Expression of E-selectin, ICAM-1 and VCAM-1 on bronchial biopsies from allergic and non-allergic asthmatic patients. Int. Arch. Allergy Immunol10619956977 |
| 14. | Ohkawara Y., Yamauchi K., Maruyama N., Hoshi H., Ohno I., Honma M., Tanno Y., Tamura G., Shirato K., Ohtani H.In situ expression of the cell adhesion molecules in bronchial tissues from asthmatics with air flow limitation: in vivo evidence of VCAM-1/VLA-4 interaction in selective eosinophil infiltration. Am. J. Respir. Cell Mol. Biol.121995412 |
| 15. | Montefort S., Lai C. K. W., Kapahi P., Leung J., Lai K. N., Chan H. S., Haskard D. O., Howarth P. H., Holgate S. T.Circulating adhesion molecules in asthma. Am. J. Respir. Crit. Care Med.149199411491152 |
| 16. | Takahashi N., Liu M. C., Proud D., Yu X.-Y., Hasegawa S., Spannhake E. W.Soluble intracellular adhesion molecule 1 in bronchoalveolar lavage fluid of allergic subjects following segmental antigen challenge. Am. J. Respir. Crit. Care Med.1501994704709 |
| 17. | De Rose V., Rollo G., Bucca C., Ghio P., Bertoletti M., Baderna P., Pozzi E.Intercellular adhesion molecule-1 is upregulated on peripheral blood T lymphocyte subsets in dual asthmatic responders. J. Clin. Invest.94199418401845 |
| 18. | Wegner C. D., Gundel R. H., Reilly P., Haynes N., Letts L. G., Rothlein R.Intercellular adhesion molecule-1 (ICAM-1) in the pathogenesis of asthma. Science2471990456459 |
| 19. | Gundel R. H., Wegner C. D., Torcellini C. A., Letts L. G.The role of intercellular adhesion molecule-1 in chronic airway inflammation. Clin. Exp. Allergy221992569575 |
| 20. | Richards, I. M., K. P. Kolbasa, G. E. Winterrowd, C. A. Hatfield, S. L. Vonderfecht, S. F. Fidler, R. L. Griffin, J. R. Brashler, R. F. Krzesicki, C. L. Lane, D. C. Anderson, L. M. Sly, N. D. Staite, and J. E. Chin. 1996. Role of intercellular adhesion molecule-1 in antigen-induced lung inflammation in Brown Norway rats. Am. J. Physiol. 271(Lung Cell. Mol. Physiol. 15):L267–L276. |
| 21. | Nakajima H., Sano H., Nishimura T., Yoshida S., Iwamoto I.Role of vascular cell adhesion molecule 1/very late activation antigen 4 and intercellular adhesion molecule 1/lymphocyte function-associated antigen 1 interactions in antigen-induced eosinophil and T cell recruitment into the tissue. J. Exp. Med.179199411451154 |
| 22. | Kennedy J. D., Hatfield C. A., Fidler S. F., Winterrowd G. E., Haas J. V., Chin J. E., Richards I. M.Phenotypic characterization of T lymphocytes emigrating into lung tissue and the airway lumen after antigen inhalation in sensitized mice. Am. J. Respir. Cell Mol. Biol.121995613623 |
| 23. | Schmid I., Schmid P., Giorgi J. V.Conversion of logarithmic channel numbers into relative linear fluorescence intensity. Cytometry91989533538 |
| 24. | Montgomery, D. C. 1984. Randomized blocks, latin squares, and related designs. In Design and Analysis of Experiments. John Wiley & Sons, Inc., New York. 123–163. |
| 25. | Chanez P., Vignola A. M., Lacoste P., Michel F. B., Godard P., Bousquet J.Increased expression of adhesion molecules (ICAM-1 and LFA-1) on alveolar macrophages from asthmatic patients. Allergy481993576580 |
| 26. | Issekutz A. C., Issekutz T. B.Monocyte migration to arthritis in the rat utilizes both CD11/CD18 and very late activation antigen-4 integrin mechanism. J. Exp. Med.181199511971203 |
| 27. | Lobb R. R., Hemler M. E.The pathophysiologic role of α4 integrins in vivo. J. Clin. Invest.94199417221728 |
| 28. | de Fougerolles A. R., Stacker S. A., Schwarting R., Springer T. A.Characterization of ICAM-2 and evidence for a second counter- receptor for LFA-1. J. Exp. Med.1741991253267 |
| 29. | de Fougerolles A. R., Springer T. A.Intercellular adhesion molecule-3, a third adhesion counter-receptor for lymphocyte function-associated molecule-1 on resting lymphocytes. J. Exp. Med.1751992185190 |
| 30. | Cabañas C., Hogg N.Ligand intercellular adhesion molecule 1 has a necessary role in activation of integrin lymphocyte function-associated molecule-1. Proc. Natl. Acad. Sci. USA90199358385842 |
| 31. | Hogg N., Harvey J., Cabanas C., Landis R. C.Control of leukocyte integrin activation. Am. Rev. Respir. Dis.1481993S55S59 |
| 32. | Lollo B. A., Chan K. W. H., Hanson E. M., Moy V. T., Brian A. A.Direct evidence for two affinity states for lymphocyte function-associated antigen-1 on activated T cells. J. Biol. Chem.26819942169321700 |
| 33. | Semnani R. T., Nutman T. B., Hochman P., Shaw S., van Seventer G. A.Costimulation by purified intercellular adhesion molecule 1 and lymphocyte function-associated antigen-3 induces distinct proliferation, cytokine and cell surface antigen profiles in human “naive” and “memory” CD4+ T cells. J. Exp. Med.180199421252135 |
| 34. | Krzesicki R. F., Winterrowd G. E., Brashler J. R., Hatfield C. A., Griffin R. L., Fidler S. F., Kolbasa K. P., Shull K. L., Richards I. M., Chin J. E.Identification of cytokine and adhesion molecule mRNA in murine lung tissue and isolated T cells and eosinophils by semi-quantitative reverse transcriptase-polymerase chain reaction. Am. J. Respir. Cell Mol. Biol.16199719 |
| 35. | Kung T. T., Stelts D. M., Zurcher J. A., Adams G. K., Egan R. W., Kreutner W., Watnick A. S., Jones H., Chapman R. W.Involvement of IL-5 in a murine model of allergic pulmonary inflammation: prophylactic and therapeutic effect of an anti-IL-5 antibody. Am. J. Respir. Cell Mol. Biol131995360365 |
| 36. | Foster P. S., Hogan S. P., Ramsay A. J., Matthaei K. I., Young I. G.Interleukin 5 deficiency abolishes eosinophilia, airways hyperreactivity, and lung damage in a mouse asthma model. J. Exp. Med.1831996195201 |
| 37. | Tomassini M., Tsicopoulos A., Tai P. C., Gruart V., Tonnel A.-B., Prin L., Capron A., Capron M.Release of granule proteins by eosinophils from allergic and nonallergic patients with eosinophilia on immunoglobulin-dependent activation. J. Allergy Clin. Immunol.881991365375 |
| 38. | Lamkhioued B., Gounni A. S., Gruart V., Pierce A., Capron A., Capron M.Human eosinophils express a receptor for secretory components. Role in secretory IgA-dependent activation. Eur. J. Immunol251995117125 |
| 39. | Monteiro R. C., Hostoffer R. W., Cooper M. D., Bonner J. R., Gartland G. L., Kubagawa H.Definition of immunoglobulin A receptors on eosinophils and their enhanced expression in allergic individuals. J. Clin. Invest.92199316811685 |
