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J Thorac Cardiovasc Surg 1995;110:15-21
© 1995 Mosby, Inc.
CARDIAC AND PULMONARY REPLACEMENT |
Kyoto, Japan
Received for publication April 19, 1994. Accepted for publication Nov. 1, 1994. Address for reprints: Hiromi Wada, MD, Department of Thoracic Surgery, Chest Disease Research Institute, Kyoto University, 53 Kawahara cho, Shogoin, Sakyo-Ku, Kyoto 606, Japan.
Abstract
Lung transplantation is now an accepted therapeutic option for patients with end-stage lung disease, and an early diagnosis of rejection is essential in the management of these patients. Adult T-cell leukemia-derived factor (ADF), known as a human homolog of thioredoxin, has been shown to be induced by a variety of stresses. In this study we examined ADF expression in lung tissues and bronchoalveolar lavage cells after canine lung transplantation to determine whether it could be induced by allogenic stimulations and could be used to diagnose early rejection. Allotransplantations were performed in adult mongrel dogs, and immunosuppression was performed from the day of operation to the fifth postoperative day. No immunosuppressant was given from the sixth to the tenth postoperative days. Animals were put to death on the tenth postoperative day. Bronchoalveolar lavage was performed on the fifth and tenth postoperative days, and the lavage cells and lung tissues were examined immunohistochemically with anti-ADF antibody. The grades of rejection were as follows: grade 1 in two animals, grade 2 in three animals, and grade 3 in two animals. The percentages of ADF high-producer cells in bronchoalveolar lavage cells on the fifth and tenth postoperative days were 4.29% ± 2.65% and 26.6% ± 3.99%, respectively (p < 0.01). The percentages of ADF high-producer cells in normal healthy dogs and in those with grade 1, grade 2, and grade 3 rejection were 3.00% ± 1.64%, 20.5% ± 9.00%, 25.5% ± 6.06%, and 34.5% ± 6.50%, respectively. The percentage in each rejection group was significantly higher than that in normal healthy dogs (p < 0.05). These results suggest that examination of bronchoalveolar lavage cells with ADF staining may be useful in the early diagnosis of rejection. (J THORAC CARDIOVASCSURG 1995;110:15-21)
Adult T-cell leukemia–derived factor (ADF) was first defined as the interleukin-2 receptor (IL-2R) 
-inducing factor produced by adult T-cell leukemia cell lines.
1,2 Complementary deoxyribonucleic acid cloning of ADF has shown a remarkable homology between ADF and Escherichia coli–derived thioredoxin, a coenzyme catalyzing the reduction of proteins in prokaryotic systems. 3-5 Thioredoxin contains a redox active disulfide (-Cys-Gly-Pro-Cys-) and has a variety of biologic activities as a hydrogen donor, including formation of deoxyribonucleotides from ribonucleotides catalyzed by ribonucleotide reductase 6 and degradation of insulin. 4 Furthermore, thioredoxin is involved in an electron-transfer system common to a variety of organisms and tissues 5 and has radical scavenging properties. 7,8 ADF expression can also be induced by a variety of stresses, including x-rays, ultraviolet irradiation, and hydrogen peroxide. 9 On the other hand, we have shown in gel retardation assays that recombinant ADF markedly enhances the binding of NF-
B to the target sequence in IL-2R chain promoter. Thus ADF is presumably required in the process of activating NF-B systems. 9 According to these findings, it was suggested that ADF expression might be induced by allogenic stimulation. In our preliminary experiments, we observed no ADF high-producer cells (ADFh cells) in transplanted canine lung tissues without rejection, and ADFh cells increased as rejection progressed. 10 We hypothesized that ADFh cells in bronchoalveolar lavage (BAL) fluid would increase in number as rejection progressed. To examine this hypothesis we performed left lung allotransplantation in adult mongrel dogs and observed the expression of ADF in biopsy specimens of the lung and in BAL cells. We also tried to determine whether analysis of cells from BAL fluid for ADF might be a useful tool in the diagnosis and monitoring of early rejection.
MATERIALS AND METHODS
Animals and anesthesia
Seven adult mongrel dogs weighing between 6 and 24 kg were used. General anesthesia was performed by the method described previously. 11-13 All animals received humane care in compliance with the "Principles of Laboratory Animal Care" formulated by the National Society for Medical Research and the "Guide for the Care and Use of Laboratory Animals" prepared by the Institute of Laboratory Animal Resources and published by the National Institutes of Health (NIH publication No 85-23, revised 1985). In addition, we tested one animal with pneumonia resistant to therapy and used five healthy dogs as negative controls.
Allotransplantation of the left lung
Allotransplantation of the left lung was performed by the method of Veith, 11 Yumazaki, 12 Yokomise, 13 and their coworkers. The warm ischemic time for the transplantation was 50 to 100 minutes.
Immunosuppression
Optimal doses of FK-506 (supplied by Fujisawa Pharmaceutical Co., Osaka, Japan) were injected intramuscularly (0.1 mg/kg per day) from the day of operation to the fifth postoperative day. 14 No immunosuppressant was administered from the sixth to the tenth postoperative days. Penicillin G was given by drip infusion on the day of operation and then aminobenzyl penicillin orally for 10 days.
BAL
A flexible fiberoptic bronchoscope (model P10, Olympus Corp., Lake Success, N.Y.) was introduced into the transplanted lung up to a subsegmental bronchus. Sterile normal saline solution (50 ml) was infused through the operating channel, and gentle aspiration was applied. This procedure was repeated once. BAL fluid was filtered through gauze to remove mucus. BAL cell differential counts were performed on Wright-Giemsa-stained smears after cytocentrifugation of 2 x 104 cells. BAL cell counts were performed with a hemocytometer on the fifth and tenth postoperative days.
Antibodies
Rabbit polyclonal antibody against ADF was raised by immunization with the ADF C-peptide conjugated with bovine serum albumin in the presence of complete Freund's adjuvant. The details of the procedure of antibody purification have been described previously. 15 Western blot analysis of recombinant ADF and dog serum showed the same 13 kd band (data not shown).
Immunohistochemical analysis of ADF
BAL cell immunohistochemistry was performed on cytocentrifugation of 2 x 104 cells on glass slides coated with poly-L-lysine. The slides were fixed initially with 50% Bouin's solution and 50% paraformaldehyde solution and then washed in phosphate-buffered saline solution. Lung tissues obtained by biopsy on the tenth postoperative day were fixed in Bouin's solution for 4 hours, embedded in paraffin, and cut into 5 µm thick sections. The slides were dewaxed in toluene and dehydrated in graded ethanol solution. Slides of cells and tissues were stained as follows: Endogenous peroxidase activity was blocked with hydrogen peroxide (0.3% in methanol for 6 minutes). Nonspecific reactions were blocked with normal goat serum. The staining procedure was performed with a DAKO LSAB Kit (DAKO, Carpinteria, Calif.). The slides were incubated with anti-ADF antibody 0.4 µg/ml for 90 minutes at 37º C or normal rabbit immunoglobulin as a negative control, then incubated with a mixture of avidin-biotin horseradish peroxidase complex, and developed with 3-amino-9-ethylcarbazol. Counterstaining was performed with hematoxylin.
Histopathology
Serial sections were stained with hematoxylin and eosin.
Assessment
The degree of rejection was classified according to "The grading of acute rejection by International Society for Heart Transplantation." 16 Two hundred BAL cells on a glass slide were counted, and the percentage of ADFh cells was determined. Cell profiles were also studied. Each histopathologic assessment was performed by two independent pathologists.
We did not perform biopsies on the fifth postoperative day because the procedure might have interfered with the experimental results
Statistical analysis
All values were expressed as the mean ± standard error. Statistical evaluation of the data was performed with Student's t test for either paired or unpaired observations. A value of less than 0.05 was accepted as significant.
RESULTS
Allograft rejection
The grades of rejection were as follows: grade 1 in two animals, grade 2 in three animals, and grade 3 in two animals. No histologic evidence of infection was found in any of the specimens. Fig. 1 (grade 2) shows many perivascular mononuclear infiltrates surrounding venules and arterioles (Fig. 1, A, hematoxylin-eosin stain). Red-stained ADFh cells can be seen mainly in the perivascular cuffing (Fig. 1, B, ADF stain).
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An early diagnosis of rejection is essential in the management of patients after lung transplantation. Open lung biopsy remains the gold standard for the diagnosis of rejection. However, the procedure is invasive and not always clinically practical. Transbronchial lung biopsy is less invasive and can be performed repeatedly. Unfortunately, inasmuch as an accurate diagnosis of rejection requires 12 to 15 biopsy specimens, 17 complications such as bleeding and pneumothorax are common. In addition, immunocompromised hosts with Pneumocystis carinii or cytomegalovirus show perivascular cuffing resembling that observed in rejected lung allografts. 18 Achterrathand coworkers 19 first advocated the use of BAL in transplanted canine lungs in 1975 and suggested that BAL might provide early clues to the diagnosis of lung allograft rejection. Since then several investigations have been reported. 20-23
In this study we especially investigated ADF expression on BAL cells on the fifth and tenth postoperative days, using normal healthy dogs as controls. Our assumption was that there would be no rejection on the fifth postoperative day because the animals were receiving immunosuppressant drugs until this time, but that by the tenth postoperative day there would be early rejection. Our pathologic data from day 10 animals indicate that this approach was valid because these animals showed early rejection. We did not perform biopsies on day 5 animals because we were concerned that this could lead to complications and adversely affect our protocol. However, other data indicate that the immunosuppression protocol we used is effective. 14 There were few ADFh cells among the BAL cells obtained from the control dogs or from animals on the fifth postoperative day when they were well immunosuppressed, but on the tenth postoperative day, when we could make a diagnosis of rejection, ADFh cells had increased significantly (control versus tenth postoperative day and fifth versus tenth postoperative day, p < 0.01). The percentage of ADFh cells in each rejection group was significantly higher than that in the control dogs (p < 0.05). This suggests that allogenic stimulation increased the number of ADFh cells in BAL fluid. In this model we found ADFh cells in the early stage of rejection--a critical finding, because to prevent serious consequences of rejection, it is necessary to start treatment in the early stages of rejection (grade 1 or 2). It took only 3 hours to perform the immunohistochemical staining, so that prompt therapy can be initiated soon after rejection is suspected. Another advantage of this approach is that it is relatively simple to apply.
ADF expression can be induced by a variety of stresses. The mechanism by which activated alveolar macrophages express ADF is not obvious, but there is much evidence that dithiol-related reducing conditions are needed for the in vitro proliferation of lymphoid cells. We have shown in gel retardation assays that recombinant ADF markedly enhances the binding of NF-B to the target sequence in IL-2R chain promoter. Thus ADF may play some role in the process of activating NF- B. 9 We hypothesize that a similar phenomenon occurs in the process of antigen presentation in alveolar macrophages under allogenic stimulation.
We determined the percentage of ADFh cells in the total BAL cell pool, because it is particularly difficult to distinguish small macrophages from medium-sized or large lymphocytes. Because we thought that most cells were macrophages, in another experiment we tried to show indirectly that ADFh cells have phagocytic activity. We incubated the BAL cells obtained on the tenth postoperative day with Zymosan treated with dog serum. Of the cells that morphologically resembled alveolar macrophages, 95% phagocytized zymosan, and the percentages of ADFh cells before and after incubation were 18% and 22%. These data indicate that ADFh cells are probably macrophages. Although the clinical usefulness of this approach appears promissory, a number of further studies are clearly required.
Of particular importance are studies to determine whether the measurement of ADFh cells can distinguish between rejection and infection, the usual problem facing clinicians. We had a chance to examine BAL cells from a dog with pneumonia. The percentage of ADFh cells was 8%. This finding suggests that measurement of ADFh cell count can help in distinguishing between rejection and infection.
In this study we could not determine whether the ADFh cells were from the donors or the recipients However, donor lymphocytes and macrophages are incompletely replaced by those of the recipient during the first 6 weeks after transplantation. 24 Human leukocyte antigen phenotyping of BAL cells or other studies are needed to identify the origin of ADFh cells correctly.
Footnotes
From the Department of Thoracic Surgery, Chest Disease Research Institute,a and the Department of Prevention and Therapeutics, Institute for Virus Research,b Kyoto University, Kyoto, Japan. 
References
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