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(Chest. 2003;124:2283-2292.)
© 2003 American College of Chest Physicians

High Expression of p40tax and Pro-inflammatory Cytokines and Chemokines in the Lungs of Human T-Lymphotropic Virus Type 1-Related Bronchopulmonary Disorders*

Yoriko Yamazato, MD; Akiko Miyazato, MD; Kazuyoshi Kawakami, MD; Satomi Yara, MD; Hiroshi Kaneshima, MD and Atsushi Saito, MD

* From the First Department of Internal Medicine (Drs. Yamazato, Miyazato, Kawakami, Yara, and Saito), Faculty of Medicine, University of the Ryukyus; and Urasoe General Hospital (Dr. Kaneshima), Okinawa, Japan.

Correspondence to: Kazuyoshi Kawakami, MD, PhD, The First Department of Internal Medicine, Faculty of Medicine, University of the Ryukyus, 207 Uehara, Nishihara, Okinawa 903-0215, Japan; e-mail: kawakami{at}med.u-ryukyu.ac.jp


   Abstract
TOP
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Study objective: Human T-lymphotropic virus type 1 (HTLV-1) is closely associated with the development of certain pulmonary diseases, such as bronchiolitis, although the pathologic mechanism remains unclear. To elucidate the pathogenesis of HTLV-1–associated bronchopulmonary disorders, we analyzed the relationship between expression of p40tax, a regulatory component of HTLV-1 that stimulates various host genes, and synthesis of pro-inflammatory cytokines and chemokines by cells in BAL fluid (BALF) obtained from HTLV-1–infected patients.

Design: Reverse transcription-polymerase chain reaction was used to compare the expression of p40tax and pro-inflammatory cytokines and chemokines messenger RNA (mRNA) in BALF of 10 HTLV-1 carriers and 7 healthy subjects. We also studied the correlation between these parameters and the proportion of lymphocytes in BALF.

Results: The expression levels of pro-inflammatory cytokines (interferon [IFN]-{gamma}, interleukin-2) and chemokines (monocyte chemotactic protein-1, macrophage inflammatory protein [MIP]-1{alpha}, IFN-{gamma}–inducible protein-10 [IP-10]) were significantly higher in BALF of patients than of healthy subjects. The expression of IFN-{gamma} and MIP-1{alpha} mRNA correlated with that of p40tax. IFN-{gamma} and IP-10 mRNA expression correlated with the proportion of lymphocytes in BALF. The percentage of lymphocytes in BALF increased with higher expression levels of p40tax mRNA, although the correlation was not significant.

Conclusion: Our results suggested that p40tax seems be involved in the development of HTLV-1–associated bronchopulmonary disorders at least in part through the local production of pro-inflammatory cytokines and chemokines.

Key Words: human T-lymphotropic virus type 1 • lung disease • p40tax • pro-inflammatory cytokines, chemokines


   Introduction
TOP
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Human T-lymphotropic virus type 1 (HTLV-1) is the etiologic agent of adult T-cell leukemia1 2 and HTLV-1–associated myelopathy/tropical spastic paraparesis (HAM/TSP).3 4 Adult T-cell leukemia is a neoplastic disease characterized by monoclonal expansion of HTLV-1–transformed cells, whereas HAM/TSP is thought to be caused by immunologic reaction to infected CD4+ T cells in the CNS. In addition, many other pathologic conditions have also been proposed to be associated with HTLV-1 infection, including arthropathy (HTLV-1–associated arthropathy),5 uveitis (HTLV-1–associated uveitis [HAU]),6 and inflammatory pulmonary diseases.7 8 9 10 11 These disorders are often found simultaneously in an individual, indicating that systemic involvement occurs during HTLV-1 infection. Sugimoto and colleagues7 8 9 10 were the first to describe pulmonary disorders in patients with HAM/TSP and HAU, which are pathologically characterized by T-lymphocytic alveolitis. Furthermore, Maruyama and co-workers12 reported similar pulmonary lesions in asymptomatic carriers of this virus. In BAL fluid (BALF) of these patients, the number of HTLV-1–infected T cells and level of viral activation are high,10 13 14 and T cells express activation markers such as interleukin (IL)-2 receptor at high levels.9 15 16 These observations suggest the possible involvement of this virus in the development of bronchopulmonary disorders.

However, a direct relationship between HTLV-1 infection and pathogenic conditions remained unsubstantiated until recently.17 Using transgenic mice expressing gene segments of HTLV-1 virus including env and pX, we demonstrated the development of inflammatory process with lymphocytic infiltration in peribronchiolar and perivascular areas and alveolar septa in the lungs. Such pathologic changes correlated well with the level of local expression of p40tax messenger RNA (mRNA) in the lungs and resembled the histopathologic findings in patients with HTLV-1 infection. The protein product of HTLV-1 pX gene, p40tax, transactivates a variety of cellular genes, including pro-inflammatory cytokines and chemokines.18 19 Our subsequent study20 with the transgenic mice provided evidence suggesting that p40tax contributed to the development of bronchopulmonary disorders through the induction of several pro-inflammatory cytokines and chemokines.

Analysis of BALF cells obtained from HTLV-1–infected individuals has shown activation of tax/rex gene, and that such activation is closely correlated with the percentage of accumulating lymphocytes.14 Further studies16 21 have shown significantly high concentrations of CC chemokines and soluble adhesion molecules in BALF of HTLV-1–infected patients compared with those of healthy control subjects. In the present investigation, we extended these earlier studies by comparing the expression of p40tax, pro- and inflammatory cytokines and chemokines mRNA levels in BALF between HTLV-1–infected patients and healthy individuals. We also evaluated the relationship between these parameters and lymphocyte counts in BALF of HTLV-1–infected subjects. Our results suggested the involvement of p40tax in the development of HTLV-1–associated pulmonary disorders at least in part through the production of pro-inflammatory cytokines and chemokines in humans.


   Materials and Methods
TOP
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Study Subjects
We examined 10 HTLV-1–seropositive individuals with lung lesions and various pulmonary symptoms (1 man and 9 women; age range, 36 to 76 years), as summarized in Table 1 . HTLV-1 seropositivity was examined using the particle agglutination methods (New Seroclit-anti-HTLV-1; Sanko; Tokyo, Japan). All patients were evaluated to be in a carrier stage. Seven of the patients had chronic bronchiolitis, one patient had hypersensitivity pneumonitis diagnosed by surgical biopsy, and two patients had suspected autoimmune diseases. Chest CT showed various abnormalities in these patients, but diffuse centrilobular nodules was a common pattern. We also investigated seven healthy individuals (four men and three women; age range, 37 to 70 years). Informed consent was obtained from all patients and healthy individuals, and the human experimentation guidelines of our institutions were followed in all clinical research protocols.


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  		Table 1.. Characteristics of HTLV-1–Infected Patients

 
BAL and Cell Preparation
BAL was performed using a standard technique. The patient was first premedicated by IM injection of atropine, 0.5 mg. After local anesthesia with 2% lidocaine, a flexible fiberoptic bronchoscope (BF-P; Olympus; Tokyo, Japan) was wedged into a subsegment of the right middle lobe for lavage. An aliquot of 50 mL of sterile physiologic saline solution at body temperature was instilled through the bronchoscope, and the fluid was immediately retrieved by gentle suction using a sterile syringe. Saline solution instillation was performed three or four times. The collected BALF was passed through two sheets of gauze and transported to the laboratory at 4°C within 30 to 60 min. After washing twice with calcium and magnesium-free phosphate-buffered saline solution supplemented with 1% heat-inactivated fetal calf serum, total cells were counted using a hemocytometer. An aliquot was then adjusted to 1 x 106 cells/mL, and a 0.1-mL sample of each cell suspension was spun down onto a glass slide at 160g for 3 min using cytocentrifuge (Auto Smear CF-12D; Sakura; Tokyo, Japan). The slides were dried, fixed, and then stained with Diff-Quick (Kokusai Shinyaku; Kobe, Japan) for determination of differential cell counts. The supernatants were stored at – 70°C until assayed. The remaining cells were pelleted by centrifugation at 400g for 10 min at 4°C, mixed with 1 mL of Isogen (Wako; Osaka, Japan), and stored at – 80°C until use. The proportions of CD4+, CD8+, and CD25+ cells were analyzed by flow cytometry (EPICS XL System II; Beckman Coulter; Fullerton, CA) after staining with fluorescein isothiocyanate-conjugated anti-CD4 or anti-CD8 monoclonal antibody (DAKO; Glostrup, Denmark) and phycoerythrin-conjugated anti-CD25 monoclonal antibody (DAKO).

Extraction of RNA and Reverse Transcription-Polymerase Chain Reaction
Total RNA of BALF cells was isolated by Isogen (Nippon Gene; Tokyo, Japan) based on the instructions provided by the manufacturer. Reverse transcription was carried out by mixing 5 µg of the sample RNA solution (15 µL) with 2 µL of hexadeoxyribonucleotide mixture (GIBCO BRL; Life Technologies; Tokyo, Japan). This solution was incubated for 2 min at 95°C and quickly cooled on ice. In the next step, 12 µL of a solution containing 6 µL of 5 x reverse transcriptase buffer (250 mM Tris-HCl [pH 8.3], 375 mM KCl, and 15 mM MgCl2) [GIBCO BRL], 0.5 µL of ribonuclease inhibitor (200 U/mL) [GIBCO BRL], 3 µL of 100 mM dithiothreitol, and 2.5 µL of 10 mM deoxynucleoside triphosphate was added, and tubes were incubated for 2 min at 37°C. The reactions were then treated with 1.0 µL of Moloney murine leukemia virus reverse transcriptase (200,000 U/mL) [GIBCO BRL] and incubated for 60 min at 37°C. After receiving 45 µL of 0.7 mol/L NaOH and 40 mM ethylenediamine tetra-acetic acid, the tubes were incubated for 10 min at 65°C and quickly cooled on ice. The resultant complementary DNA was precipitated with 75% ethanol overnight at – 70°C. The precipitates were washed once with 75% ethanol, dried, and resuspended in 50 µL of diethyl pyrocarbonate-treated distilled water. The samples were stored at – 20°C until use.

Polymerase chain reaction (PCR) was carried out in an automatic DNA thermal cycler (Perkin-Elmer Cetus; Norwalk, CT). We added 1.0 µL of the sample complementary DNA solution to 49 µL of the reaction mixture, which contained 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, 10 µg/mL of gelatin, deoxynucleoside triphosphate (each at a concentration of 200 µM), 1.0 µM sense and anti-sense primers, and 1.25 U of Ampli-Taq DNA polymerase (Takara Shuzo; Kyoto, Japan). For detection of p40tax mRNA, the complementary DNA was subjected to two-step amplification using nested primer pairs of the second splice junction site of tax/rex mRNA.14 22 The reaction mixture including 1.0 µM outer primers (RPX-11: 5'-TAA TAG CCG CCA GTG GAA AG; and pX-9: TGA TCT GAT GCT CTG GAC AG) was then subjected to 30 cycles of amplification. Subsequently, a 1-µL aliquot of the first-step PCR reaction mixture was added to 49 µL of a PCR cocktail containing inner primers (RPX 3: 5'-ATC CCG TGG AGA CTC CTC AA; and RPX 4: AAC ACG TAG ACT GGG TAT CC) and subjected to another 38 cycles. In each cycle of the first and second PCRs, the mixture was subjected to denaturation at 94°C for 2 min, annealing at 60°C for 2 min and extension at 72°C for 2 min. The primers used for PCR detection of pro-inflammatory cytokine and chemokine mRNAs are listed in Table 2 . The preparations in the microtubes were amplified by using a three-temperature PCR system usually consisting of denaturation at 94°C for 30 s, primer annealing at 55°C for 30 s and extension at 72°C for 2 min for glyceraldehyde 3-phosphate dehydrogenase (GAPDH), monocyte chemotactic protein (MCP)-1, macrophage inflammatory protein (MIP)-1{alpha}, MIP-1ß, and interferon (IFN)-{gamma}–inducible protein-10 (IP-10). PCR conditions of denaturing at 94°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 30 s were used for IL-2 and IFN-{gamma}. The number of amplification cycles was determined for samples not reaching the amplification plateau. The cycle numbers were 23, 30, 26, 28, 24, and 25 for GAPDH, IFN-{gamma}, MCP-1, MIP-1{alpha}, MIP-1ß, and IP-10, respectively. For detection of IL-2 mRNA, a nested PCR was conducted at 30 cycles for the first step and 10 cycles for the second step. The PCR products of GAPDH and a particular molecule were electrophoresed on the identical 2% agarose gel, stained with 0.5 µg/mL of ethidium bromide, and observed with ultraviolet transilluminator. The obtained bands of amplified DNA were quantified using NIH Image Analysis Software (version 1.61; National Institutes of Health; Bethesda, MD), and the result of a particular molecule was expressed as a relative amount as compared to that of GAPDH.


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  		Table 2.. Primer Sequences

 
Statistical Analysis
Statistical analysis was performed using a Mann-Whitney U test. The linear least-squares regression analysis was used to estimate the relationship between two different parameters; p < 0.05 was considered significant.


   Results
TOP
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Characteristics of BALF Cells
Table 3 summarizes the results BALF cell analysis in HTLV-1–infected patients and healthy control subjects. Neither leukemic cells nor pathogens associated with opportunistic infection were found in BALF of these patients. In HTLV-1–infected patients, the mean total cell count was 5.7 x 105/mL (range, 0.8 to 12.7 x 105/mL) and lymphocyte proportion was 23.4% (range, 0.6 to 53.9%; > 20% in four patients). The proportion of neutrophils ranged from 0 to 59.5%, with a mean value of 17.0%. It should be noted that 6 of 10 patients had high alveolar neutrophilic counts. The ratio of CD4+/CD8+ cells was inverted in 8 of 10 patients (mean, 1.67; range, 0.27 to 6.92). In healthy control subjects, the mean values of total cell counts and proportion of lymphocytes were 0.8 x 105/mL (range, 0.4 to 1.4 x 105/mL) and 8.9% (range, 5.3 to 11.8%), respectively. The above values were significantly higher in HTLV-1–infected patients than in healthy control subjects.


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  		Table 3.. Results of BALF Analysis*

 
Detection of p40tax mRNA and Its Correlation With Lymphocytes in BALF of HTLV-1–Infected Patients
To examine the replication of HTLV-1, we amplified HTLV-1 p40tax mRNA by nested reverse transcription-PCR (RT-PCR). In all but two HTLV-1–infected patients (patient 7 and patient 10), positive bands of p40tax mRNA were detected at the second PCR, although the bands were very faint in patient 2 and patient 8 (Fig 1 ). The obtained bands of amplified DNA were quantified using the NIH Image Analysis Software and expressed relative to that of GAPDH. As shown in Figure 2 , the percentage of lymphocytes increased with higher expression of p40tax mRNA in BALF of HTLV-1–infected patients; however, the correlation between the two parameters was not significant.



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  		Figure 1.. Amplification and detection of p40tax and GAPDH mRNAs in BALF cells of HTLV-1–seropositive individuals. Expected size of products from p40tax (145 base-pair) and GAPDH (508 base-pair) are indicated. All but patients 7 and 10 expressed p40tax mRNA. M = molecular size marker.

 


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  		Figure 2.. The expression level of p40tax mRNA was calculated relative to that of GAPDH. The linear least-squares regression analysis was used to estimate the relationship between p40tax mRNA and the percentage of lymphocytes in BALF. NS = not significant.

 
Production of Pro-inflammatory Cytokine and Chemokine mRNAs by BALF Cells of HTLV-1–Infected Patients
HTLV-1 p40tax is known to activate a variety of cellular genes, including pro-inflammatory cytokines1 and chemokines,18 as well as its own replication. In the next series of experiments, we determined the mechanism underlying the accumulation of lymphocytes by examining the production of pro-inflammatory cytokines (IL-2 and IFN-{gamma}) and chemokines (MCP-1, MIP-1{alpha}, MIP-1ß, and IP-10) by BALF cells of HTLV-1–infected patients and healthy subjects at mRNA level using RT-PCR. The results were expressed as the relative value of each band to that of GAPDH. As shown in Figure 3 , the expression levels of IL-2, IFN-{gamma}, MCP-1, MIP-1{alpha}, and IP-10 mRNAs in HTLV-1 carriers were significantly higher than in healthy subjects. In HTLV-1-infected patients with positive p40tax expression, similar difference was found also in MIP-1ß (data not shown).



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  		Figure 3.. Comparison of messenger RNA expression of pro-inflammatory cytokines (top, A) and chemokines (bottom, B) in BALF cells by semiquantitative RT-PCR between HTLV-1 carriers and healthy control subjects. The results are expressed relative to GAPDH. See Figure 2 legend for expansion of abbreviation.

 
Relationship Between Expression of p40tax mRNA and Pro-inflammatory Cytokines and Chemokines
Based on the given results, we examined the relationship between p40tax mRNA expression and that of pro-inflammatory cytokines and chemokines in BALF of HTLV-1–infected patients. There was a significant relationship between p40tax mRNA expression and that of IFN-{gamma} (r = 0.663, p < 0.05) and MIP-1{alpha} (r = 0.749, p < 0.05). However, the expression of other pro-inflammatory cytokines and chemokines did not correlate with that of p40tax (Fig 4 ).



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  		Figure 4.. Correlation between mRNA expression of p40tax and various pro-inflammatory cytokines (top, A) and chemokines (bottom, B) in the BALF cells of HTLV-1 carriers. The results of RT-PCR analysis are expressed relative to GAPDH. Data were analyzed using the linear least-squares regression analysis. See Figure 2 legend for expansion of abbreviation.

 
Relationship Between Expression of Pro-inflammatory Cytokines/Chemokines and Lymphocytes in BALF
Finally, to elucidate the involvement of these pro-inflammatory cytokines and chemokines in lymphocytic accumulation in the lungs, we evaluated the relationship between their expression and proportion of lymphocytes in BALF of HTLV-1–infected patients. As shown in Figure 5 , there was a significant correlation between the expression of IFN-{gamma} and IP-10 mRNAs and percentages of lymphocytes (r = 0.864, p < 0.01 for IFN-{gamma}; r = 0.770, p < 0.01 for IP-10). However, there was no significant correlation between percentages of lymphocytes in BALF and other pro-inflammatory cytokines and chemokines tested in this study.



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  		Figure 5.. Correlation between mRNA expression of pro-inflammatory cytokines (top, A) and chemokines (bottom, B) and proportion of lymphocytes in BALF of HTLV-1 carriers. The results of RT-PCR analysis are expressed relative GAPDH. Data were analyzed using the linear least-squares regression analysis. See Figure 2 legend for expansion of abbreviation.

 

   Discussion
TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Clinical and epidemiologic studies7 8 9 10 11 12 have established that HTLV-1 is closely associated with certain bronchopulmonary disorders. Accumulation of lymphocytes and activated T cells, as well as abundant HTLV-1–infected T cells and viral activity, have been demonstrated in BALF of HTLV-1–infected patients.8 9 10 11 12 13 14 15 16 25 Although HTLV-1 was suggested to be involved in bronchopulmonary lesions, characterized by lymphocytic accumulation, the precise mechanism of such response is not fully understood. In the present study, we analyzed BALF cells in eight HTLV-1 carriers with pulmonary symptoms. The expression of p40tax mRNA was detected in all but two patients. BALF of these patients contained a high proportion of lymphocytes, which correlated with the level of p40tax mRNA expression in the lungs. Consistent with previous findings18 19 that p40tax transactivated many cellular genes, including cytokines and chemokines, our results demonstrated high expression of pro-inflammatory cytokines (IFN-{gamma} and IL-2) and chemokines (MCP-1, MIP-1{alpha}, MIP-1ß, and IP-10) at mRNA level in BALF cells of HTLV-1–infected patients, compared with healthy individuals. Furthermore, there was a significant correlation between expression of p40tax mRNA and that of MIP-1{alpha} and also between expression of IFN-{gamma} and IP-10 mRNA and proportion of lymphocytes in BALF of HTLV-1–infected patients. Furthermore, the expression levels of other pro-inflammatory cytokines and chemokines tended to increase with increased expression of p40tax mRNA and proportion of lymphocytes in BALF. Thus, our study suggested that the activation of p40tax gene might be involved in the development of bronchopulmonary lesions through induction of pro-inflammatory cytokines and chemokines.

To characterize pulmonary involvement in HTLV-1 carriers, lung specimens were obtained from 32 HTLV-1–positive Japanese patients with diffuse pulmonary infiltrates.26 Unlike patients with HAM/TSP and HAU,7 9 10 11 these patients had respiratory symptoms, such as cough, sputum, and dyspnea, and showed abnormalities in pulmonary function tests. Among that group, 14 patients manifested chronic bronchiolitis, pathologically characterized by lymphocytic inflammation of small bronchi and membranous bronchioles with or without fibrotic changes in surrounding lung parenchyma.26 In agreement with the above reports, all patients in the present study had clinical symptoms; based on chest CT findings, six of eight patients were suspected of having bronchiolitis. The expression of p40tax mRNA was detected in all patients, except for two cases. One patient was suspected of having bronchiolitis based on open-lung biopsy, and recovered from clinical symptoms such as cough and dyspnea on effort after cessation of cigarette smoking. Thus, the diagnosis in this case was made as respiratory bronchiolitis, rather than HTLV-1–associated lung disease.

Several investigations have demonstrated that chemokines play a central role in the development of cellular inflammatory lesions with accumulating leukocytes27 28 and that pro-inflammatory cytokines regulate the production of certain chemokines.29 30 For example, IFN-{gamma} induces MCP-1, MIP-1{alpha}, MIP-1ß, and IP-10 production.31 32 33 Chemokines are classified into four groups (CXC, CC, CX3C, and C) according to the position of the first two cysteins.34 While glutamate-leucine-arginine (ELR)+ CXC chemokines, such as IL-8, cause the trafficking of neutrophils, CC chemokines, including MCP-1, MIP-1{alpha}, and MIP-1ß, mainly attract monocytes and lymphocytes. However, IP-10, a CXC chemokine lacking the ELR sequence, is secreted by IFN-{gamma}-stimulated macrophages, endothelial cells, fibroblasts, and keratinocytes, and selectively attracts activated CD4+ T cells.33 34 35 36 It should be noted that mononuclear leukocytes are the major cell populations that accumulate in the involved organs of HTLV-1–associated disorders.7 8 9 10 11 Compatible with this notion, our results showed high levels of mononuclear cell-trafficking CC and ELR- CXC chemokines in BALF of HTLV-1 carriers with bronchopulmonary lesions, compared with control subjects. Furthermore, the synthesis of these chemokines largely correlated with the proportion of BALF lymphocytes in HTLV-1–infected patients. Thus, our data suggest that some chemokines may be involved in the pathogenesis of mononuclear leukocyte-mediated inflammatory lesions in the lung caused by HTLV-1 infection.

HTLV-1–infected cells or p40tax gene-transfected cells produce various types of pro-inflammatory cytokines, such as IL-1{alpha}, IL-1ß, IL-2, IL-6, IFN-{gamma}, tumor necrosis factor-{alpha}, and transforming growth factor-ß, and chemokines, such as IL-8, IP-10, and MIP-1{alpha}.18 Previous studies17 20 suggested that activation of the p40tax gene might contribute to the development of bronchopulmonary lesions in HTLV-1–infected patients through the induction of pro-inflammatory cytokines and chemokines. Our data confirmed these conclusions by demonstrating the correlation of p40tax gene expression with the expression of pro-inflammatory cytokines and chemokines and also with the proportion of lymphocytes in BALF. Other studies from our laboratory in transgenic mice bearing the pX region of this virus demonstrated high expression levels of pro-inflammatory cytokines, IL-1ß, tumor necrosis factor-{alpha}, and IFN-{gamma}, and chemokines, MCP-1, RANTES (regulated upon activation, normal T-cell expressed and secreted), MIP-1{alpha}, and IP-10, in the lungs of these mice, which showed lymphocytic infiltration in the peribronchiolar and perivascular areas and alveolar septa.17 20 Moreover, there was a significant relationship between expression of p40tax and MCP-1 mRNA, and the expression of RANTES and IP-10 mRNAs correlated well with the severity of lung lesions in these mice. These findings obtained from animal studies17 20 supported the above hypothesis raised in the present study. Similarly, Higashiyama et al14 demonstrated activation of tax/rex gene, which was proportional to the extent of lymphocytosis in BALF of HTLV-1–infected patients. Subsequent studies21 by the same group indicated increased synthesis of CC chemokines at a protein level in BALF of the same patients and a significant correlation between MIP-1{alpha} concentration and percentage of activated T cells in BALF.

Bronchopulmonary disorders associated with HTLV-1 are characterized by lymphocyte accumulation in BALF.7 8 9 10 11 12 16 21 However, in the present study, the proportion of neutrophils was also high in HTLV-1–infected patients with respiratory symptoms. Neutrophil accumulation is considered as evidence of bacterial infection. However, when patients suffer from bacterial infection, BAL is always conducted after complete resolution of such infection, ie, following improvement of clinical symptoms and normalization of serum C-reactive protein levels and peripheral blood WBC counts, by appropriate treatment with antibiotics. For these reasons, the relative increase of neutrophils in BALF does not mean the presence of complicated bacterial infection. In one study,18 p40tax enhanced the expression of IL-8 in addition to other pro-inflammatory cytokines and chemokines. Consistent with this finding, IL-8 mRNA was expressed at a higher level in patients with BALF neutrophilia than in control subjects (data not shown). Thus, we believe that IL-8, induced by p40tax activation independent of bacterial infection, may contribute to the recruitment of neutrophils into the lung lesions of HTLV-1–infected patients.

In conclusion, our present study first provided the evidence in support of the involvement of various pro-inflammatory cytokines and chemokines transactivated by p40tax in the development of HTLV-1–associated bronchopulmonary disorders. However, our understanding of the precise mechanism underlying these processes remains incomplete. Further investigations are necessary to determine the exact cell type that contributes to the expression of p40tax in human lungs and the precise mechanism that discriminates patients with pulmonary lesions from healthy carriers.


   Acknowledgements
 
The authors thank Dr. F. G. Issa (Word-Medex; Sydney, Australia) for reading and editing the manuscript.


   Footnotes
 
Abbreviations: BALF = BAL fluid; ELR = glutamate-leucine-arginine; GAPDH = glyceraldehyde 3-phosphate dehydrogenase; HAM/TSP = human T-lymphotropic virus type 1–associated myelopathy/tropical spastic paraparesis; HAU = human T-lymphotropic virus type 1-associated uveitis; HTLV-1 = human T-lymphotropic virus type 1; IFN = interferon; IL = interleukin; IP-10 = interferon-{gamma}-inducible protein-10; MCP = monocyte chemotactic protein; MIP = macrophage inflammatory protein; mRNA = messenger RNA; PCR = polymerase chain reaction; RT-PCR = reverse transcription-polymerase chin reaction

Received for publication July 11, 2002. Accepted for publication June 18, 2003.


   References
TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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