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The Journal of Immunology, 2003, 170: 5712-5718.
Copyright © 2003 by The American Association of Immunologists

T Cells of Atopic Asthmatics Preferentially Infiltrate Into Human Bronchial Xenografts in SCID Mice 1

Kaori Tsumori*, Hirotsugu Kohrogi2,*, Eisuke Goto*, Naomi Hirata*, Susumu Hirosako*, Kazuhiko Fujii*, Makoto Ando*, Osamu Kawano* and Hiroshi Mizuta{dagger}

* First Department of Internal Medicine and {dagger} Department of Orthopedic Surgery, Kumamoto University School of Medicine, Kumamoto, Japan


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
T cells play an important role in the pathogenesis of bronchial asthma. However, it is not completely known how circulating lymphocytes infiltrate into the airways of asthmatic patients. Because SCID mice are unable to reject xenogenic transplants, many xenotransplant models using various human tissues have been developed. Therefore, to examine the interaction between bronchi and T lymphocytes of asthma, it may be possible to use the human bronchial xenograft and PBMC xenograft in SCID mice. We transplanted human bronchi into the subcutaneum of SCID mice and i.p. injected PBMCs that were obtained from patients with atopic asthma, atopic dermatitis and rheumatoid arthritis, and normal subjects (asthmatic, dermatitis, rheumatic, and normal huPBMC-SCID mice). There was no difference in the percentage of CD3-, CD4-, CD8-, CD25-, CD45RO-, CD103-, and cutaneous lymphocyte Ag-positive cells in PBMCs among the patients with asthma, dermatitis, rheumatoid arthritis, and normal subjects, and CD3-positive cells in peripheral blood of asthmatic, dermatitis, rheumatic, and normal huPBMC-SCID mice. The number of CD3-, CD4-, and CD8-positive cells in the xenografts of asthmatic huPBMC-SCID mice was higher than those of dermatitis, rheumatic, and normal huPBMC-SCID mice. IL-4 mRNA and IL-5 mRNA were significantly higher in the xenografts of asthmatic huPBMC-SCID mice than those in the xenografts of normal huPBMC-SCID mice, but there were no significant differences in the expressions of IL-2 mRNA or IFN-{gamma} mRNA between them. These findings suggest that T cells, especially Th2-type T cells, of asthmatics preferentially infiltrate into the human bronchi.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Asthma is characterized by airway inflammation, airway hyperresponsiveness, and reversible airflow limitation. Many inflammatory cells are participating in the pathogenesis of asthma. Among these cells, T cells play a pivotal role in orchestrating airway inflammation. In asthmatic airways, it was reported that 1) the higher number of T cells in adults (1) and children (2) and CD45-, CD3-, CD4-, and CD8-positive cells were found in groups with asthma (3), although activated eosinophils are also increased; 2) lymphocytes (PBMCs) of corticosteroid-resistant asthma are not sensitive to corticosteroid in inhibiting the proliferation and production of IL-2 and IFN-{gamma} but are sensitive to cyclosporin A (4). T cells have an important role in sensitization and immune responses. They release IL-3, -4, and -5 and GM-CSF, a pattern compatible with predominant activation of the Th2-type cell population (5), suggesting that activated T cells may contribute to cause inflammation with eosinophil accumulation during allergen-induced asthma (6). Similar pathologic changes in the bronchus were also observed in nonatopic asthma (7). Recently, it was reported that Th2-type cells infiltrated and reduced expression of Th1-type cell transcription factor, T-bet, which is associatedwith asthma (8). These findings suggest that Th2-type cells may play an important role in the pathogenesis of asthma.

The processes where inflammatory cells infiltrate into the inflamed tissues are explained by a multistep paradigm including rolling, activation, firm adhesion, and diapedesis (9, 10). Some adhesion molecules, chemokines, and their counterreceptors are known to be involved in these processes (11, 12, 13). We previously reported the expressions of adhesion molecules E-selectin, VCAM-1, and ICAM-1 in the human bronchial vasculature by allergen-induced mast cell activation (14). These findings suggested that allergic response induced by IgE and mast cells causes preparation for infiltration of lymphocytes, eosinophils, and neutrophils into the bronchial mucosa. In other organs, e.g., intestine, skin, and lymph nodes, tissue-specific adhesion molecules, chemokines, and their receptors that recruit T cells have already been discovered (15, 16, 17, 18, 19). Despite these discoveries, it is not well known how T cells recirculate into the bronchial mucosa in allergic diseases such as asthma.

One of the strategies for treating asthma is to block the infiltration of T cells into the bronchi. Because SCID mice are unable to reject xenogenic transplants, many xenotransplant models using various human tissues have been developed (20, 21, 22, 23) and we have demonstrated that human bronchial tissues can be transplanted and maintained well in the subcutaneum of the SCID mice (24). To study the mechanism of recruitment of T cells to the human bronchi, we create a novel human-SCID mice chimera model by transplanting human bronchi and human PBMCs derived from patients with asthma, atopic dermatitis and rheumatoid arthritis, and normal subjects (we arbitrarily called them asthmatic huPBMC-SCID mice, dermatitis huPBMC-SCID mice, rheumatic huPBMC-SCID mice, and normal huPBMC-SCID mice). In the present study, we found that T cells derived from asthmatic patients preferentially recruited into the human bronchial xenografts in the PBMC-SCID mice. These T cells in the xenografts showed the Th2-type cytokine-secreting pattern. These results partly explained that T cells in asthmatic patients preferentially recirculate into the bronchial tissues.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Tissue preparation

Human bronchial tissues were obtained from 19 patients undergoing surgery for lung cancer. The patients did not have complaints suggesting underlying pulmonary diseases, including chronic bronchitis, asthma and emphysema, and their chest roentgenograms were normal except for lung cancer. Their pulmonary functions (vital capacity and forced expiratory volume in 1 s) were within normal limits. Immediately after the lung or lobe was resected, the tissues were placed in Krebs-Henseleit solution. Airway segments from the third- to fifth-generation bronchi, which were macroscopically normal, were cut into 10-mm lengths as bronchial rings. All tissues were prepared within 2 h after the resection.

PBMC preparation and cell analysis

PBMCs were isolated with Ficoll-Paque from heparinized blood samples collected from 19 patients with atopic asthma, 6 with atopic dermatitis, 5 with rheumatoid arthritis, and 9 normal subjects. Asthma was diagnosed by episodic symptoms of airflow obstruction and the significant reversibility shown by an increase of >12% and a 200-ml increase in forced expiratory volume in 1 s (25). Atopy was diagnosed by increased levels of serum IgE CapRAST against house dust or Dermatophagoides pteronyssinus and Dermatophagoides farinae to >2+. Atopic dermatitis was diagnosed by dermatologists and rheumatoid arthritis was diagnosed by rheumatologists according to the American Rheumatism Association 1987 revised criteria for the classification of rheumatoid arthritis. None of the patients were receiving oral corticosteroids. Patients with asthma were receiving inhaled corticosteroid (beclomethasone: <800 µg/day), patients with atopic dermatitis were receiving corticosteroid ointments as needed, and patients with rheumatoid arthritis were receiving nonsteroidal anti-inflammatory drugs. Thus, it was highly possible that there was no systemic effect of corticosteroids.

Part of PBMCs were analyzed using a FACScan flow cytometer (BD Biosciences, Mountain View, CA) and the positive rate for each surface marker was calculated using CellQuest software (BD Biosciences). Anti-CD3 (UCHT1; DAKO, Glostrup, Denmark), anti-CD4 (MT310; DAKO), anti-CD8 (DK25; DAKO), anti-CD19 (HD37; DAKO), anti-CD25 (ACT-1; DAKO), anti-CD45RO (UCHL1; BD PharMingen, San Diego,CA), anti-CD103 (2G5; Immunotech, Marseille, France), anti-cutaneous lymphocyte Ag (CLA)3 (HECA-452; BD PharMingen), and FITC-conjugated rabbit anti-mouse Ig (DAKO) were used for flow cytometry.

Xenotransplantation of human bronchi and PBMCs

As described previously (24), at day 0, after anesthetizing 5- to 10-wk-old SCID mice (CLEA Corporation, Tokyo, Japan) with ethyl ether, we made one s.c. tunnel using a 10-mm incision in the abdomen, and the prepared bronchial tissues were inserted into the tunnel. On day 7, 2 x 107 PBMCs from patients with atopic asthma, atopic dermatitis, rheumatoid arthritis, or from normal subjects were injected into the peritoneal cavities of the SCID (huPBMC-SCID) mice. On day 21, the mice were sacrificed, and the xenografts and the tissues of the mice were harvested and placed in optimal cutting temperature embedding medium, snap-frozen in liquid nitrogen, and stored at -80°C until cryostat sectioning. Peripheral blood leukocytes of sacrificed mice were isolated using an ammonium-chloride-potassium buffer and analyzed with flow cytometry using anti-human CD3 (UCHT1; DAKO) and FITC-conjugated rabbit anti-mouse Ig (DAKO).

Immunohistochemical staining

The frozen tissues were cut 6-µm thick and were stained immunohistochemically as described previously (14). The Abs and reagents used for staining were anti-CD3 (UCHT1; DAKO), -CD4 (MT310; DAKO), -CD8 (DK25; DAKO), -CD25 (ACT-1; DAKO), -CD45RO (UCHL1; BD PharMingen), -CD103 (2G5; Immunotech), and -CLA (HECA-452; BD PharMingen). For a peroxidase-dependent brown color reaction, 3,3'-diaminobenzidine (Dojin, Kumamoto, Japan) was used as the substrate. In the peroxidase-dependent staining, the tissues were counterstained with Mayer’s hematoxylin.

Microscopic assessment and quantification of lymphocytes

The stained sections were examined with a VANOX AHBS3 microscope (Olympus, Tokyo, Japan). We counted lymphocytes that were positively stained with the respective Abs in the lamina propria and we expressed the number of T cells in the lamina propria per millimeter-squared. Counting of stained sections was initially done without knowledge of the xenografts of asthmatic, dermatitis, rheumatic, and normal huPBMC-SCID mice.

RT-PCR analysis

Total cellular RNA was extracted from the freshly isolated bronchi, the bronchial xenografts of asthmatic and normal huPBMC-SCID mice, and from PBMCs of patients with asthmatics and normal subjects, using a MicroFast Track 2.0 kit (Invitrogen, Carlsbad, CA), and reverse-transcribed at 37°C for 50 min in 20 µl containing 200 U Moloney murine leukemia virus reverse transcriptase (Life Technologies, Gaithersburg, MD), 10 mM DTT, 1 mM each of dATP, dTTP, deoxycytidine triphosphate (dCTP), and deoxyguanidine triphosphate (dGTP); and 0.5 µg oligo-dT primer (Life Technologies). Reactions were stopped by heat inactivation for 15 min at 70°C. Sequences were amplified from cDNA by PCR using specific primers for IL-2, IFN-{gamma}, IL-4, IL-5, and {beta}-actin. The primer sequences were as follows: IL-2, sense, 5-caagaatcccaaactcaccagg-3, antisense, 5-caatggttgctgtctcatcagc-3; IFN-{gamma}, sense, 5-ctcttggctgttactgccag-3, antisense, 5-tccttgatggtctccacact-3; IL-4, sense, 5-cggacacaagtgcgatatcacc-3, antisense, 5-ccaacgtactctggttggcttcc-3; IL-5, sense, 5-cgaactctgctgatagccaatg-3, antisense, 5-ccactcggtgttcattacaccaag-3; {beta}-actin, sense, 5-ccagccatgtacgttgct-3, antisense, 5-cttctccagggaggagct-3. PCR amplifications were performed in 50 µl containing 2 µl of cDNA, 25 pmol of each primer, 1.5 mM MgCl2, 1.25 U Taq DNA polymerase (Applied Biosystems, Foster City, CA). PCR of {beta}-actin was performed by 30 cycles of 60 s at 94°C, 120 s at 60°C and 120 s at 72°C. PCR of IL-2, IL-4, and IL-5 were performed by 35 cycles of 60 s at 94°C, 80 s at 60°C, and 70 s at 72°C. PCR of IFN-{gamma} was performed by 35 cycles of 60 s at 94°C, 120 s at 50°C, and 120 s at 72°C. The PCR products were analyzed by electrophoresis on 1.5% agarose gels and stained with ethidium bromide. The intensity of the bands for cytokine and {beta}-actin RT-PCR products was determined by densitometry (Hoefer Scientific Instruments, San Francisco, CA). Results were expressed for each cytokine product as the ratio relative to {beta}-actin product.

Statistical analysis

Values are expressed as means ± SEM. The Mann-Whitney U test was used to analyze statistical differences. A value of p < 0.05 was taken as statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Characteristics of PBMCs of patients with asthma, atopic dermatitis, rheumatoid arthritis, and normal subjects

There was no significant difference in the percentage of CD3-, CD4-, CD8-, CD19-, CD25-, CD45RO-, and CD103-positive cells in PBMCs among patients with asthma, atopic dermatitis, rheumatoid arthritis, and normal subjects (Table I).


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  		Table I. Characteristics of PBMCsa

 
CD3 in peripheral blood of asthmatic, dermatitis, rheumatic, and normal huPBMC-SCID mice

There were no differences in the percentage of CD3-positive cells in the peripheral blood of asthmatic huPBMC-SCID mice, dermatitis huPBMC-SCID mice, rheumatic huPBMC-SCID mice, and normal huPBMC-SCID mice (5.14 ± 1.87, 6.20 ± 1.44, 6.84 ± 1.33, 4.93 ± 2.65%, respectively).

CD3-, CD4-, and CD8-positive cells in human bronchial tissue xenografts of asthmatic, dermatitis, rheumatic, and normal huPBMC-SCID mice

The number of CD3-, CD4-, and CD8-positive cells in the xenografts of asthmatic huPBMC-SCID mice was higher than that of normal huPBMC-SCID mice. There was no difference in the number of CD3-, CD4-, and CD8-positive cells between the xenografts of normal huPBMC-SCID mice and those without human PBMCs (Figs. 1, 2, and 3).



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  		FIGURE 1. Immunohistochemical staining of the xenografts of asthmatic, dermatitis, rheumatic, and normal huPBMC-SCID mice. Seven days after the transplantation of human bronchial tissue in the subcutaneum of the SCID mice, PBMCs from atopic asthmatic patients or normal subjects were injected into the peritoneal cavities of the SCID mice. We arbitrarily called them asthmatic, dermatitis, rheumatic, and normal huPBMC-SCID mice. Two weeks later, the xenografts were harvested. A, C, E, G, and I show the staining of xenografts of asthmatic huPBMC-SCID mice and B, D, F, H, and J show the staining of xenografts of normal huPBMC-SCID mice. The number of CD3- (A and B), CD4- (C and D), CD8- (E and F), CD45RO- (G and H), CD103- (I and J) positive cells are higher in the xenografts of asthmatic huPBMC-SCID mice than those of normal huPBMC-SCID mice. K and L show CD3 staining of the xenografts of dermatitis and rheumatic huPBMC-SCID mice, indicating a lower number of CD3-positive cells than that of asthmatic huPBMC-SCID mice. Bar = 100 µm; arrowheads show CD103-positive cells.

 


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  		FIGURE 2. CD3-positive cells in the xenografts of asthmatic, atopic dermatitis, rheumatoid arthritis, and normal huPBMC-SCID mice. The number of CD3-positive cells in the lamina propria of the xenografts of asthmatic huPBMC-SCID mice (asthmatic) was significantly higher than that of atopic dermatitis, rheumatoid arthritis, and normal huPBMC-SCID mice and without human PBMCs (dermatitis, rheumatic, normal) and that without PBMC injection (PBMC(-)). Each value is the mean ± SEM; n = 12, 6, 5, 9, 5 for each experiment. *, p < 0.05.

 


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  		FIGURE 3. CD4- and CD8-positive cells in the xenografts of asthmatic huPBMC-SCID and normal huPBMC-SCID mice. The numbers of CD4-positive (A) and CD8-positive cells (B) in the lamina propria of the xenografts in asthmatic huPBMC-SCID mice (asthmatic) were significantly higher than that of atopic dermatitis, rheumatic, and normal huPBMC-SCID mice (dermatitis, rheumatic, normal) and that without PBMC injection (PBMC(-)). Each value is the mean ± SEM; n = 12, 6, 5, 9, 5 for each experiment. *, p < 0.05.

 
In addition, the number of CD3-, CD4-, and CD8-positive cells in the xenografts of asthmatic huPBMC-SCID mice was higher than that of dermatitis and rheumatic huPBMC-SCID mice (Figs. 1–3). From these results, we analyzed asthmatic huPBMC-SCID mice and normal huPBMC-SCID mice in the following study.

CD25-, CD45RO-, CD103-, CD19-, and CLA-positive cells in human bronchial tissue xenografts of asthmatic and normal huPBMC-SCID mice

The ratio of CD25/CD3-positive cells in the xenografts of asthmatic huPBMC-SCID mice was higher than that without human PBMCs (9.9 ± 1.5 vs 3.2 ± 2.2, p < 0.05), but there was no significant difference in the ratio of CD25/CD3-positive cells between the xenografts of asthmatic huPBMC-SCID mice and those of normal huPBMC-SCID mice (9.9 ± 1.5 vs 9.0 ± 2.0).

The ratios of CD45RO/CD3- and CD103/CD3-positive cells in the xenografts of asthmatic huPBMC-SCID mice were higher than those of normal huPBMC-SCID mice or those of SCID mice without human PBMCs. However, there were no significant differences in the ratios of CD45RO/CD3- and CD103/CD3-positive cells between the xenografts of normal huPBMC-SCID mice or those without human PBMCs (Figs. 1 and 4).



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  		FIGURE 4. CD45RO- and CD103-positive cells in the xenografts of asthmatic huPBMC-SCID and normal huPBMC-SCID mice. The numbers of CD45RO-positive cells (A) and CD103-positive cells (B) in the lamina propria of the xenografts in asthmatic huPBMC-SCID mice (asthmatic) were significantly higher than that of normal huPBMC-SCID mice (normal) and that without PBMC injection (PBMC(-)). Each value is the mean ± SEM; n = 12, 9, 5 for each experiment. *, p < 0.05.

 
CD19-positive or CLA-positive cells were not detected in the lamina propria of either xenografts of asthmatic huPBMC-SCID mice or normal huPBMC-SCID mice (data not shown).

Expression of mRNA in PBMCs of asthmatic patients and normal subjects

The ratios of cytokine mRNA/{beta}-actin mRNA were not significantly different between asthmatics and normal subjects (Fig. 5).



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  		FIGURE 5. mRNAs of IL-4, IL-5, IL-2, and IFN-{gamma} in PBMCs of asthmatic patients and normal subjects. A and B, The ratios of their cytokine mRNA/{beta}-actin mRNA were not significantly different between asthmatics and normal subjects. C, A representative mRNA detected by RT-PCR. Each value is the mean ± SE; n = 5 for each experiment. *, p < 0.05.

 
Expression of mRNA in bronchi before transplantation and in xenografts of asthmatic and normal huPBMC-SCID mice

In the freshly isolated bronchi before transplantation, mRNA of IL-2, IL-4, IL-5, and IFN-{gamma} was not detected (data not shown). The ratios of IL-4 mRNA/{beta}-actin mRNA and IL-5 mRNA/{beta}-actin mRNA were significantly higher in the xenografts of asthmatic huPBMC-SCID mice than those in the xenografts of normal huPBMC-SCID mice (Fig. 6). mRNA expressions of IL-2 and IFN-{gamma} in the xenografts of asthmatic huPBMC-SCID mice and those of normal huPBMC-SCID mice were detected, but there were no significant differences in the cytokine mRNA/{beta}-actin mRNA between them (Fig. 6).



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  		FIGURE 6. mRNA in the xenografts of asthmatic and normal huPBMC-SCID mice. A, The ratios of IL-4 mRNA/{beta}-actin mRNA and IL-5 mRNA/{beta}-actin mRNA were significantly higher in the xenografts of asthmatic huPBMC-SCID mice than that in the xenografts of normal huPBMC-SCID mice. There were no significant differences in IL-2 mRNA/{beta}-actin mRNA and IFN-{gamma} mRNA/{beta}-actin mRNA between the xenografts of asthmatic huPBMC-SCID mice and that of normal huPBMC-SCID mice. B, A representative mRNA detected by RT-PCR. Each value is the mean ± SEM; n = 5 for each experiment. *, p < 0.05.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We found that numbers of CD3-, CD4-, and CD8-positive cells infiltrated into the human bronchial xenografts that were transplanted in the subcutaneum of asthmatic huPBMC-SCID mice, but not in those of dermatitis, rheumatoid, and normal huPBMC-SCID mice. Furthermore, we found that IL-4 mRNA and IL-5 mRNA in the xenografts of asthmatic huPBMC-SCID mice were expressed.

It is known that lymphocytes, especially Th2-type lymphocytes, infiltrate into the airway tissue in asthmatics (5, 6). However, the mechanism of infiltration is not well-known. It is difficult to perform bronchial biopsies many times to examine the inflammatory cell infiltration after Ag challenge in asthmatics. Therefore, it is necessary to have animal models of bronchial asthma to study the mechanism of inflammatory cell infiltration into the airway tissue. However, no good animal models of asthma are currently available. The present model using SCID mice, in which human bronchial tissues and asthmatic PBMCs were transplanted, may be useful for studying the infiltrating mechanism of lymphocytes from blood vessels to bronchial tissues.

The numbers of CD3-, CD4-, and CD8-positive cells in the xenografts of asthmatic huPBMC-SCID mice were higher than those of dermatitis, rheumatic, and normal huPBMC-SCID mice and those of SCID mice without PBMCs. In the infiltrated CD3-positive cells in the xenografts of asthmatic huPBMC-SCID mice, the number of CD4-positive cells was higher than that of CD8-positive cells (p < 0.05), and CD45RO-positive cells infiltrated into the xenografts. There was no significant difference in the percentage of CD3-, CD19-, CD25-, CD45RO-, and CD103-positive cells in PBMCs among patients with asthma, atopic dermatitis, rheumatoid arthritis, and normal subjects, and there were no differences in the number of human CD3-positive cells in PBMCs among asthmatic, dermatitis, rheumatic, and normal huPBMC-SCID mice. Thus, these results suggested that CD3-, CD4-, and CD8-positive cells of asthmatics preferentially infiltrated into the human bronchial xenografts from blood vessels. We previously found that in the human bronchial xenografts without human PBMC transfer, human bronchial intraepithelial lymphocytes survive for >5 mo, while the T cells in the lamina propria decreased rapidly within 1 mo (24). Thus, in the present model, the T cells in the lamina propria mainly originated from the peripheral blood of SCID mice in which human PBMCs were transferred.

In the present model, donors of bronchial xenografts and PBMCs were allogenic. So, it was possible that the infiltration of T cells into the xenografts was due to rejection of the allogenic bronchial tissues. However, the number of CD3-, CD4-, and CD8-positive cells that infiltrated into the xenografts of asthmatic huPBMC-SCID mice was higher than those of dermatitis, rheumatic, and normal huPBMC-SCID mice. Furthermore, expressions of IL-4 mRNA and IL-5 mRNA were higher in the bronchial xenografts of asthmatic huPBMC-SCID mice than those of normal huPBMC-SCID mice. Thus, there were differences in the number and the function of infiltrating lymphocytes in the xenografts between asthmatic and normal huPBMC-SCID mice. Therefore, the present results suggested that the infiltration of T cells into the xenografts was mainly specific to asthma. Despite the significant infiltration of CD3-, CD4-, and CD8-positive cells into the xenografts of asthmatic huPBMC-SCID mice, a small number of CD3-, CD4-, and CD8-positive cells were found in the xenografts of dermatitis, rheumatic, and normal huPBMC-SCID mice, and even in the xenografts of the mice without injection of PBMCs. Possible explanations for these phenomena were 1) resident T cells of the xenografts, 2) recognition of allografts, and/or 3) the homing effect into the bronchial tissues due to previous airway diseases such as infectious acute bronchitis.

Expressions of IL-4 mRNA and IL-5 mRNA were higher in the xenografts of asthmatic huPBMC-SCID mice than those of normal huPBMC-SCID mice, while they were not different in the PBMCs between asthmatic and normal huPBMC-SCID mice. In the xenografts, IL-4-producing cells may be CD4-positive cells, basophils, and mast cells. In the xenografts of normal huPBMC-SCID mice, IL-4 mRNA was not detectable. Therefore, resident mast cells in the xenografts may not be the origin of IL-4 mRNA. After allergen challenge in the asthmatic bronchi, basophils produce IL-4 (26). Because, in the present study, we have not studied the immunohistochemical staining for basophils, a role of basophils in this model should be investigated in the future. Thus, it is suggested that asthmatic peripheral blood, probably T cells, infiltrated from vascular beds into the bronchial xenografts and that they started producing IL-4 and IL-5.

Lymphocytes homing from blood into organs is mediated by a series of sequential interactions between the lymphocytes and the vascular endothelium in specialized postcapillary venules. Tissue specificity is characterized by the use of different combinations of adhesion and chemoattractant receptors at distinct organs such as the skin and intestines, but it is not well-clarified at the lungs and bronchi. In inflamed skin, T cells express CLA and P-selectin ligands, and vascular endothelium expresses E-selectin and P-selectin (27). In addition, CCR4 and thymus- and activation-regulated chemokine (TARC) are important in the recognition of skin vasculature by circulating T cells and in directing lymphocytes to the skin (28). In contrast, in the intestine, mucosal addressin cell adhesion molecule-1 is expressed on postcapillary venules in the intestinal lamina propria where T cells that highly express {alpha}4{beta}7 integrin are migrated. Furthermore, these T cells express CCR9, which is the specific receptor for the chemokine thymus-expressed chemokine, that are expressed by small intestinal epithelium (29).

In contrast to the skin and intestine, adhesion molecules and chemokines for T cell homing into the airways and lungs was not determined. Panina-Bordignon et al. (30) demonstrated that, in asthmatics after allergen challenge, T cells in the bronchi express IL-4, CCR4, and CCR8, and that bronchial epithelia express the CCR4-specific ligands monocyte-derived chemokine (MDC) and TARC. These findings suggested that allergic response in the asthmatic airways causes release of MDC and TARC in the epithelium which stimulates Th2-type cells through CCR4 to migrate into the airway tissue where allergic inflammation is ongoing. In contrast, in the present model, IL-4 and IL-5 mRNA were detected in the xenografts of the asthmatic huPBMC-SCID mice, but there were no significant differences in the expressions of CCR4, CCR8, MDC, and TARC in the xenografts between asthmatic and normal huPBMC-SCID mice (data not shown). The reason for this gap may be due to the stimulation of the airways by allergen. We did not stimulate the airways by Ag while Panina-Bordignon et al. (30) did. In fact, their findings showed that the expressions of IL-4, CCR4, and CCR8 were not observed before allergen exposure to the asthmatics. Thus, the expressions of IL-4, CCR4, and CCR8 may require allergen stimulation in the inflamed region. In addition, TARC and MDC are expressed not only in airway epithelium but also in the other organs such as skin (31, 32) and the nose (33). Taken together, it is suggested that T cells in the peripheral blood, especially Th2-type cells, of asthmatic patients migrate from blood into bronchial tissue without inflammatory stimulation by a mechanism other than chemokines such as MDC and TARC and their ligands of CCR4. In the present study, CD103-positive cells, known to be {alpha}E{beta}7 integrin, were highly infiltrated into the xenografts of the mice injected with asthmatic PBMCs compared with those injected with PBMCs of normal subjects. {alpha}E{beta}7 integrin-positive cells were found in nasal polyps and intraepithelial T cells (34). The present findings suggested that {alpha}E{beta}7 integrin-positive PBMCs preferentially infiltrated into the xenografts, or that a part of asthmatic PBMCs infiltrated into the xenografts and then expressed {alpha}E{beta}7 in the xenografts. To treat airway inflammation in asthmatics further an investigation is needed to find specific adhesion molecules, chemokines, and their receptors which regulate the infiltration of T cells into the airways.

In summary, CD3-, CD4-, CD8-, CD45RO-, and CD103-positive cells of asthmatic patients preferentially infiltrated into the human bronchial xenografts in the SCID mice, and IL-4 mRNA and IL-5 mRNA were expressed in the xenografts of the mice in which asthmatic PBMCs were transferred. These results suggest that T cells, especially Th2-type lymphocytes, of asthmatic patients selectively infiltrate into bronchial tissues and release Th2-type cytokines to cause chronic eosinophilic bronchial inflammation.


   Acknowledgments
 
We thank Drs. Fujino and Saishoji of Kumamoto Chuoh Hospital, Dr. Baba of Kumamoto City Hospital, Dr. Johno of Dermatology in Kumamoto University Hospital, and Dr. Tsukano of Kumamoto Orthopedic Hospital for their kind cooperation in providing human lung tissues. We also thank the entire staff of the First Department of Internal Medicine and Department of Orthopedic Surgery of Kumamoto University School of Medicine.


   Footnotes
 
1 This study was supported by Scientific Grants-in-Aid for Scientific Research ((C)12670564) from the Ministry of Education, Science and Culture of Japan. Back

2 Address correspondence and reprint requests to Dr. Hirotsugu Kohrogi, First Department of Internal Medicine, Kumamoto University School of Medicine, 1-1-1 Honjo, Kumamoto 860-8556, Japan. E-mail address: kohrogi{at}kaiju.medic.kumamoto-u.ac.jp Back

3 Abbreviations used in this paper: CLA, cutaneous lymphocyte Ag; TARC, thymus- and activation-regulated chemokine; MDC, monocyte-derived chemokine. Back

Received for publication May 17, 2002. Accepted for publication March 20, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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