Spontaneous Proliferative Response of Peripheral Blood Mononuclear Cells in Vitro as an Indicator of High Response to Human T-Lymphotropic Virus Type 1 Infection in an Infant With Interstitial Pneumonia

Human T-lymphotropic virus type 1 (HTLV-1) is the causative virus in adult T cell leukemia (ATL)1 and HTLV-1-associated myelopathy2/tropical spastic paraparesis3(HAM/TSP). Characteristic immunologic findings accompanied with HTLV-1 infection have been observed among ATL and HAM/TSP patients. When peripheral blood mononuclear cells (PBMC) are cultured in vitro with or without purified HTLV-1 virion antigen, a response of T cells with HTLV-1 antigen is similarly observed among ATL patients, HAM/TSP patients and normal controls.4 Meanwhile the T cell response without HTLV antigen is different in three groups; ATL patients show spontaneous proliferative response (SPR) of PBMC but their response is weak, while most HAM/TSP patients include relatively high and low responders, and normal controls rarely show SPR.4 SPR of lymphocytes has been also observed among ∼50% of asymptomatic individuals infected with HTLV-1.5 This SPR has been suggested to be a response to interleukin 2 (IL-2) through the expression on IL-2 receptors by the px gene of HTLV-1.6 These findings have been observed among HTLV-1-infected individuals in adulthood,4, 5 but there is no information for the SPR of lymphocytes among children, although there are HTLV-1-infected children. We report the first infant infected with HTLV-1 who had interstitial pneumonia (IP) and a high SPR of PBMC. Case report. A 3-year-old boy complained of a cough and tachypnea, which gradually exacerbated and was accompanied by cyanosis and growth failure. In March, 1989, at 4 years of age, he was admitted to the Kagoshima University Hospital. After normal delivery, the subject was fed breast milk for 12 months. He developed sinusitis at 3 years of age. He had no history of allergic diseases, blood transfusion or surgery. Nobody in his family had ATL, other HTLV-1-associated diseases, allergic diseases or IP. On admission the patient's weight was 11.7 kg (−2.2 SD), and height was 94.7 cm (−1.4 SD). Tachycardia (124 beats/min) and tachypnea (80 breaths/min) were noted and cyanosis at rest appeared in his nail beds. Breath sounds were diminished but neither audible crackles nor heart murmurs could be heard. The enlarged liver was palpable 3 cm below the right costal margin, but splenomegaly or systemic lymphadenopathy was not detected. Neurologic evaluation was normal. The patient's chest roentgenogram showed bilateral diffuse nodular infiltrates with ill-defined margins that tended to fuse. There was no lymphadenopathy in the hilum. Laboratory values were as follows: leukocyte count was 11.9 × 103 cells/l of 59% neutrophils without increased immature forms, 33% lymphocytes, 3% monocytes and 5% eosinophils. No leukemic cells with ATL cells were found. C-reactive protein was negative. Blood chemistry findings showed an elevated value of total protein of 8.1 g/dl and lactate dehydrogenase of 586 IU. Serum immunoelectrophoresis disclosed an increase in the gamma-globulin of 20.4% with increases in IgG of 2153 mg/dl and increases in IgA of 265 mg/dl. Alpha-1-antitrypsin and angiotensin-converting enzyme were normal. Arterial blood gases on room air compared at rest and exercise showed a decrease of arterial oxygen tension, 76.1 mm Hg, 53.4 mm Hg, respectively, and an increase of alveolar-arterial oxygen difference, 21.8 mm Hg, 52.0 mm Hg, respectively, but no increase of carbon dioxide tension, 41.8 mm Hg, 35.6 mm Hg, respectively. The patient's peripheral blood tested positive for HTLV-1 antibody by the particle agglutination test (Fujirebio, Tokyo, Japan), and the titer was at the dilution of 1:4096. After his antibody isotypes were confirmed as IgG and IgM by Western blotting (Eisai, Tokyo, Japan), his family were also examined and his parents and paternal and maternal grandmothers also tested positive for HTLV-1 antibody. Bacteriologic, acid-fast and fungus cultures of sputum showed no pathogenic agents. The antibodies of influenza A and B viruses, parainfluenza viruses, cytomegalovirus, Epstein-Barr virus, adenovirus and Mycoplasma did not disclose increase of titers in the paired examinations. Antibody testing against HIV was also negative by the particle agglutination test(Fujirebio, Tokyo, Japan). An open lung biopsy was performed to confirm the diagnosis and to determine the steroid medication prescribed after informed consent was obtained from his parents. It demonstrated interstitial aggregation of moderate infiltrating lymphocytes with lymphoid follicles, small numbers of plasma cells, eosinophils and granulomas. Interstitial fibrosis was also present. A few lymphocytes with nuclear configuration flower-like cells were observed. There were no bacilli, fungi or Pneumocystis carinii and there was no proliferation of histiocytes. Moreover no pathologic finding of viral infection such as inclusion bodies was found. These pathologic findings confirmed the diagnosis as IP. Therefore steroid medication was started, and the patient's dyspnea and abnormal chest radiographic findings markedly improved, but his symptoms and chest radiographic findings recurred at 7 years of age after discontinuing the steroid therapy for 1 1/2 years. His peripheral lymphocytes showed remarkable increase in CD4+CD25+ cells of 9.8% of 7380 lymphocytes/μl. No infectious agents except HTLV-1 were detected at the time of recurrence of IP. Methods. The analysis of HTLV-1 tax gene expression was done by in situ hybridization as previously described.7 SK-45, a synthetic probe of single-stranded 40-base oligonucleotides of the tax region of HTLV-1 was purchased from Perkin Elmer Cetus (Norwalk, CT). Briefly, after pretreatment and prehybridization, the specimens from the patient's lung lesion which were stored in paraffin were hybridized by adding 35 pM digoxigenated SK-45 probes to the prehybridization solution for 12 h at 37°C. Hybridization products were then visualized with a nucleic acid detection kit (Boehringer Mannheim). PBMC were separated from heparinized peripheral blood of subjects. After being washed the cells were suspended in RPMI 1640 supplemented with 10% fetal calf serum, cell numbers were counted and cells were resuspended at 5 × 105 cells/ml in RPMI 1640 supplemented with 10% fetal calf serum. Each PBMC was cultured under two conditions as follows: Condition 1, at a concentration of 5 × 104 cells/well (200 μl) with phytohemagglutinin(PHA) and concanavalin A (Con A) as stimulants in triplicate at 37°C in a 5% CO2 incubator; Condition 2, in the same condition as Condition 1) without adding PHA and Con A. After 6 days of incubation, [3H]thymidine(0.1 μCi) was added to each culture, and the cells were harvested on glass fiber filters after an additional 8 h of incubation. Then radioactivity was measured by a scintillation counter (cpm). Controls comprised three boys matched to the patient for age, whose informed consent was obtained from their parents. HLA typing for A, B, C, DR and DQ antigens was performed by a standard National Institutes of Health microcytotoxicity test using the selected sera standardized according to the criteria of the 11th International Histocompatibility Workshop.8 HLA typing was performed not only for the patient but also for his grandmothers, parents and two younger siblings with informed consent of his parents and grandmothers. Informed consent was obtained from the parents of the child who served as the subject of the investigation. The present study has been reviewed by the ethics committee of Department of Pediatrics, Kagoshima University, and has been performed in accordance with the ethical standards laid down in an appropriate version (the 1989 Declaration of Hong Kong) of the 1964 Declaration of Helsinki. Results.In situ hybridization showed positive staining of HTLV-1 tax messenger RNA in the nuclei and cytoplasm of the infiltrated mononuclear cells. The proportion of positively stained-cells was ∼10% among total mononuclear cells of the lung specimens. The PBMC of the patient and his mother spontaneously increased without stimulation by PHA and Con A (Table 1). Therefore the stimulation indexes of the case and his mother was lower than those of controls. The case carried A24Cw1B54DR8DQ1 and A24Cw-B51DR9DQ3. One of his haplotypes was carried by each of his two siblings. The haplotypes of his father were close to those of his mother: A24Cw1B54DR8DQ1 and A24Cw1B61DR4DQ4 in his father; A24Cw-B51DR9DQ3 and A24Cw1B54DR4DQ4 in his mother.TABLE 1: Comparison of the proliferative response of peripheral blood mononuclear cells in vitro with or without the stimulation by PHA and Con A between the patient, his mother and three controls Discussion. Our case showed a high SPR of PBMC, commonly observed among HAM/TSP patients and some HTLV-1 carriers.4, 5, 9 This SPR, the so-called "autologous proliferative response," is not caused by oncogenic findings like ATL but by immunologic response. It is suggested that CD4+ cells of HAM/TSP patients are automatically activated through the IL-2/IL-2 receptor by the px gene of HTLV-1.6 The activation of IL-2/IL-2 receptor presents as an increase of CD25+ cells. This increase of CD4+CD25+ cells was concordantly observed in this case. Furthermore the high particle agglutination titer against anti-HTLV-1 antibody, which has often been shown among HAM/TSP patients, was present in the present case.4 Sonoda et al.10 have suggested that HTLV-1/II infection and the associated diseases might be determined by immunogenetic factors segregated with HLA alleles and haplotypes. The HLA typing for A (A24) and C (Cw1, Cw-) loci of the present case was partially the same as HAM-associated HLA haplotypes, but the haplotypes were not completely concordant to HAM-associated ones.4, 10 The SPR of lymphocytes has been observed among HTLV-1-infected individuals in adulthood,4, 5, 9 but never in childhood, because HTLV-1-associated diseases are rare in children. Our case showed new evidence that a high SPR could be observed in childhood. The present case was histologically confirmed as having IP at open lung biopsy and was distinguished from sarcoidosis or hypersensitivity pneumonitis by the clinical course and the results of laboratory examination. In this case the characteristic clinical findings of acute infectious diseases were not observed. Because virus isolation was not made, chronic viral infection can not be completely discounted from the cause of IP. However, clinical course, histologic findings and paired tests of the antibodies provided no evidence of specific infectious diseases, such as cytomegalovirus, EBV and HIV infections. Moreover no systemic diseases, such as collagen diseases and immunodeficiency diseases, accompanied this case. Pulmonary involvement in ATL is often observed as opportunistic infections and bleeding.11 Hence pulmonary involvement in HAM/TSP is suggested to be associated with its immunologic response.12-14 In the present case moderate lymphocyte infiltration, including ∼10% HTLV-1-infected cells, was detected from the lung lesion. The proportion of these infiltrated HTLV-1-infected cells was higher than that of HTLV-1-infected cells in peripheral blood among asymptomatic HTLV-1 carriers.7 It is difficult to confirm the causal relationship between HTLV-1 infection and interstitial pneumonia in the present case because the immunologic evidence of the lung lesion was not sufficient. However, there is still a possibility that an enhanced immunologic response with high SPR might play some role in developing interstitial pneumonia. Acknowledgments. We thank Dr. Yoshinori Kawabata, Research Institute of Tuberculosis, Japan Anti-tuberculosis Association, Tokyo, and Dr. Jyoji Wakimoto and Dr. Shinichiro Mori, National Sanatorium Minami-Kyusyu Hospital, Kagoshima, for the histologic examination and for their suggestions; and Dr. Syozou Nagamatsu, Kagoshima Children Hospital, Kagoshima, for his helpful advice. Furthermore we acknowledge Dr. Kazuo Tajima, Aichi Cancer Center Research Institute, Nagoya, and Professors Syunro Sonoda and Koichiro Miyata, Kagoshima University, for their critical reviews of the manuscript. Tomoko Takezaki, M.D. Mitsuru Setoyama, M.D. Toshiro Takezaki, M.D. Syuji Takei, M.D. Shinji Yashiki, B.S. Kazunobu Sueyoshi, M.D. Hidetomo Fukunaga, M.D.