JOURNAL OF PATHOLOGY, VOL.
161: 105-1 12 (1 990)
THE HISTOLOGICAL CHANGES IN TRANSBRONCHIAL BIOPSY AFTER TREATMENT OF ACUTE LUNG REJECTION IN HEART-LUNG TRANSPLANTS COLIN A. CLELLAND, TIMOTHY W. HIGENBOTTAM, SUSAN STEWART, JOHN P. SCOTT AND JOHN WALLWORK
The Heart-Lung Transplant Research Unit, Papworth Hospital, Papworth Everard, Cambridge CB3 8RE. U.K. Received 28 December 1989 Accepted 8 January 1990
SUMMARY Transbronchial lung biopsies taken during episodes of acute lung rejection in heart-lung transplant patients were examined histologically. When the diagnosis was confirmed by microscopy, the patients were treated with augmented immunosuppression by high-dose corticosteroids. A second biopsy was obtained an average of 23.5 days after commencement of treatment. These biopsies were examined to determine the histological changes caused by treatment. In most cases, there were both quantitative and morphological differences between the infiltrates in acute rejection and in the remaining perivascular infiltrates after treatment. In acute rejection, 26 of 28 biopsies contained perivascular lymphocytic infiltrates, lymphocytes being large and blast-like. Although 20 of 28 follow-up biopsies still contained perivascular infiltrates, they were smaller and the lymphocytes smaller in size. Half the biopsies in rejection contained neutrophils, but less than half contained eosinophils in the perivascular infiltrates. After treatment, all these cells were less numerous. Another feature of treated rejection was the presence of haemosiderin around vessels suggesting earlier acute vascular injury. However, haemosiderin persists long after the cellular infiltrate has disappeared and cannot be considered a reliable feature of recently treated acute lung rejection. The bronchiolar infiltrates showed a similar pattern of responses to the perivascular infiltrates. KEY woms-Heart-lung
transplantation, acute lung rejection, transbronchial lung biopsy, augmented immunosuppression.
INTRODUCTION T h e histopathology of acute lung rejection (ALR) in human heart-lung transplants (HLTs) has been described in studies o f transbronchial lung biopsies (TBBs) and post-mortem material.14 The use of the larger alligator forceps has provided good-sized TBBs, enabling the distinction to be made between ALR and pulmonary infection. Pyroninophilic, perivascular lymphocytic infiltrates are characteristic of ALR and are often associated with similar lymphocytic infiltration o f bronchioles which, in the absence o f infection, has also been attributed to ALR. Examination of TBBs, together with analysis of the clinical course of patients after HLT, has Addressee for correspondence: Dr T. W. Higenbottam, Department of Respiratory Physiology, Papworth Hospital, Papworth, Everard, Cambridge CB3 8RE,U.K.
0022-341 7/90/060101-08 $05.00 0 1990 by John Wiley & Sons, Ltd.
Table I-Histological classification of acute lung allograft rejection based on extent of inflammatory infiltration identified on TBB Grade
Histological appearances
0
No significant inflammation. Normal biopsy
1
Small, infrequent perivascular infiltrates with or without bronchiolar lymphocytic infiltrates
2
Larger, more frequent perivascular lymphocytic infiltrates with or without moderate bronchiolar lymphocytic inflammation. Occasional neutrophils and eosinophils
3
Extension of infiltrates into alveolar septa and alveolar spaces with or without bronchiolar mucosal ulceration
106
C. A. CLELLAND ET AL.
Table 11-Pre-operative diagnoses and mean ages of the 21 patients
Pre-operative diagnosis Eisenmenger’s syndrome Cystic fibrosis Pulmonary vascular disease Primary pulmonary hypertension Cryptogenic fibrosing alveolitis Sarcoidosis Churg-Strauss syndrome and pulmonary hypertension
No. of patients 8 7 1 2 1 1 1
Mean age in years
(SD) 29.6 (9.4) 22.7 (1-4) 22 23 (11) 53 43 34
Fig. 1-Transbronchial lung biopsies takenduringacutelung rejection (ALR)and aftertreatment in one patient. (a)In ALR, thereis a dense infiltrate of large lymphocytes around a small blood vessel. No neutrophils are seen. (b) After treatment, the penvascular infiltrate is smaller and the lymphocytesare reduced in size. Dark pigment can be seen even on haematoxylin and eosin staining
allowed a simple grading of histopathological severity of rejection to be devised (Table I) which helps to predict o ~ t c o r n e . ~ In the course of these studies, it has become clear that treatment, in particular augmented immunosuppression in the form of ‘pulsed’ steroids, modifies the histopathological appearance of ALR.
Corticosteroids are known to reduce lymphocytic as well as other cellular infiltrations of allo enic organ grafts both in man6 and in animals! Frequent episodes of acute rejection are also associated with an increased frequency of submucosal fibrosis and vascular sclerosis on TBB.’ Both histological features are also seen in those patients who have
LUNG HISTOLOGY AFTER TREATMENT OF REJECTION 2a
surgical technique, lung preservation system, and maintenance immunosuppressive treatment have been previously described in detail.”.” The patients were selected as each had experienced at least one episode of lung rejection. Methods of lung physi~logical”*’~ and chest radiological14 surveillance of these patients have been described earlier, together with clinical indications for TBB. The technique of TBB in HLT has been described,’ but in this study at least three biopsies were taken from each lobe of one lung (lingula being considered as a separate lobe). Processing and staining of the biopsies are now standard.’ Acute lung rejection in each patient was treated with 0-5-1 g methyl prednisolone intravenously daily for 3 days, and then oral prednisolone 1 mg/ kg/day in decreasing doses down to 10 mg daily over 10 days. Repeat TBBs were then performed, on average 23.5 days after diagnosis of ALR ( & 13,4s.D.; range: 7-58 days). Episodes of infection were excluded from the analysis. These were diagnosed by histology’ and by viral, bacterial, and fungal culture of TBB, concomitant bronchoalveolar lavage specimens, and sputa.
SIZE OF INFILTRATES
CELLS WIDE I 2 20 I I
i
ACUTE REJECTION
2b %
40
;
TREATED REJECTION
FREQUENCY
BIOPSIES 35%
107
35%
ANALYSIS
..
=
FREOUENCY OF INFILTRATES
ACUTE REJECTION
.*
TREATED REJECTION
Fig. 2-Size of perivascular infiltrates, expressed as number of cells wide (divided by 2), in ALR and TR (a) and the frequency of perivascular infiltrates around parenchymal vessels (-, none;
occasional; + +, several; and
+ + +, frequent infiltrates) (b)
The 28 pretreatment and 28 post-treatment TBBs were examined retrospectively by C.C., who had no knowledge of whether the biopsies were before or after treatment. The size and frequency of dense pyroninophilic lymphocytic perivascular infiltrates were assessed, Size was determined according to the
+, width of the infiltrate, measured by the number of
developed disabling or fatal obliterative bronchiolitis (OB).9 Here we report a study of 21 HLT patients who experienced 28 episodes of ALR. In each case, pulmonary infection was excluded by histopathology and culture. A comparison was made in each person between the histopathological appearance of TBB during ALR and TBB performed after ‘pulsed’ steroid therapy. PATIENTS AND METHODS Twenty-one of the 69 HLT patients from this institute were studied. They underwent surgery between July 1984 and July 1989. Their original diagnoses and ages are shown in Table 11. The
cells across the greatest diameter of the largest infiltrate of each biopsy. By convention, to give a measure of radius, this value was divided by two. The frequency of infiltrates was recorded semiquantitatively: none (-), occasional (+), frequent (+ +), or many (+ + +). Morphologically similar infiltration was sought in the bronchioles. The number of each type of inflammatory cell was also assessed semi-quantitatively using a scoring system of again none (-), occasional (+), or many (+ +). This scoring of cellularity was performed separately for infiltrates around parenchymal blood vessels and those around bronchioles. The presence of haemosiderin in these sites was similarly quantified. All the biopsies were allocated to a grade of severity using our recently reported classification system.’ Evidence of fibrosis was also recorded: (-) when there was none; (+) when
108
C. A. CLELLAND ETAL.
Table 111-Mean size of perivascular infiltrates,and numbers of different cell types in perivascular and bronchiolar infiltrates during ALR and after treatment. Estimates of haemosiderin deposition around parenchymal vessels and bronchioles are also included Acute rejection Mean SD
Treated rejection Mean SD
Perivascular infiltrates
Size of lymphocytic
7.1
(4.8)
infiltrate (cells wide -2) No. of lymphocytes (+’s) No. of plasma cells No. of neutrophils No. of eosinophils Amount of haemosiderin
1.32 0.11 0.61 0.50 0.61
(0.66) (0.31) (0.67) (0.63) (0.72)
1.18 0.29 0.39 0.32 0.25
(0.76) (0.45) (0.56) (0-47) (0.51)
3.8
(3.5)
0.75
(0.51) (0.31) (0.62) (0.54) (0.79)
0.11
0-39 0.18 0.86
Bronchiolar infiltrates (+ ’s)
No. of lymphocytes No. of plasma cells No. of neutrophils No. of eosinophils Amount of haemosiderin
0.93 0.29 0.32 0-11
0.61
(0.88) (0.52) (0.47) (0-41) (0.77)
the follow-up biopsies were free of perivascular lymphocytic infiltrates. There was no difference in the numbers of plasma cells before and after treatment and this cell type was only infrequently seen. There was a tendency for neutrophils to be reduced in numbers after treatment. A reduction in the small numbers of eosinophils present during ALR was RESULTS also seen after treatment. The cellular infiltrates The initial biopsies during ALR had infiltrates, affecting the bronchioles were similar on semiwhether perivascular or bronchiolar, of large blast- quantitative analysis to the perivascular infiltrates like lymphocytes with occasional neutrophils and (Table 111). There was less change in the response to eosinophils. After treatment, the lymphocytic infil- treatment than with perivascular infiltrates. Howtrates consisted mainly of small lymphocytes which ever, the trend of the changes in cell content was were more widely dispersed (Fig. 1). broadly similar. There was a slight increase in perivascular and Perivascular infiltrates were larger in ALR than in the paired follow-up biopsies in 21 of the 28 bronchiolar haemosiderin after treatment (Fig. 4). instances, the same size on four occasions, and As expected, because the basis of the histological smaller in three instances (Fig. 2a). The mean and classificationsystem rests on the intensity and extent standard deviation ( & SD) of the size of the infil- of lymphocytic infiltration of the biopsies the reductrates for the ALR and treated rejection (TR) tion in histological grade paralleled the changes groups are shown in Table 111. There was a reduc- found in the semi-quantitative analysis of perivastion in the frequency of infiltrates after treatment cular lymphocytes. The higher grades were affected (Fig. 2b). However, many follow-up biopsies still more by treatment than the lower grades (Fig. 5). contained either ‘occasional’or ‘several’ perivascu- The follow-up showed an increase in the number of lar infiltrates. Only 11 per cent of TBBs were free of biopsies with the mildest degree of fibrosis (intrainfiltrates during ALR as compared with nearly 30 alveolar and mild alveoIar septa1 fibrosis) (Fig. 5). per cent after treatment. No increase in severe fibrosis (submucosal bronAugmented immunosuppression diminished the chiolar fibrosis or obliterative airway fibrosis) was number of lymphocytes(Fig. 3). Nearly one-third of detected during this short-term period of follow-up. there was mild parenchymal fibrosis; (+ +) when parenchymal fibrosis was more extensive,and where there was bronchiolar submucosal fibrosis; and (+ + +) when there was biopsy evidence of fibrous obliteration of bronchioles.
109
LUNG HISTOLOGY AFTER TREATMENT OF REJECTION PLASMA CELLS
LYMPHOCYTES BIOPSIES
OF BIOPSIES
80
100
68%
70
60 60 40
30 20 10
..
..
0
OUANTITY OF PLASMA CELLS
QUANTITY OF LYMPHOCYTES ACUTE REJECTION
ACUTE REJECTION
TREATED REJECTION
FOSINOPHILS
NFlJTROPYlLS X BIOPSIES
X BIOPSIES 80
TREATED REJECTION
100
7
-
7%
QUANTITY OF NEUTROPHILS ACUTE REJECTION
..
..
TREATED REJECTION
7%
CIUANTITY OF EOSINOPHILS -ACUTE
REJECTION
TREATED REJECTION
Fig. %Bar charts to show assessment of the numbers of lymphocytes, plasma cells, neutrophils, and eosinophils in perivascular infiltrates in AR and TR (-, none; + ,few; + +, many)
these cell types were often reduced in numbers after treatment. One patient had persistent perivascular This paper extends our earlier studies on the infiltrates of eosinophils, but she had received HLT histology of ALR as seen on TBB.’**Paired TBBs for Churg-Strauss syndrome in association with were compared in each episode of ALR with those pulmonary hypertension. It is possible that this after completion of a course of augmented immuno- finding was related to recurrence of the primary suppressive treatment after rigorously excluding disease. On analysing the histological grade of ALR, it episodes of infection. This has enabled us to provide the first description of the histopathology of was apparent that many follow-up biopsies showed residual evidence of ALR in that small perivasular resolving rejection of the lung in man. Perivascular lymphocytic infiltrates were much infiltrates were still present. It can be questioned more frequent and larger in ALR than after treat- whether these patients should receive a further ment. Morphologically, the infiltrating lympho- course of ‘pulsed’steroids until the biopsies become cytes were smaller in size after treatment. Plasma clear of rejection. At resent, we cannot answer this. cells were infrequently observed, supporting the In an earlier study, patients who had predicted view that ALR is mainly a cell-mediated response. values for lung function and who had no respiratory Only in half the episodes of ALR were neutrophils symptoms or signs but had evidence of acute rejecof eosinophils found in significant numbers around tion on histology of TBB remained well despite no blood vessels. When present in the initial biopsy, treatment and had no more frequent rejection over
DISCUSSION
P
110
C. A. CLELLAND ET AL. 4a
PERIVASCULAR HAEMOSIDERIN
% BIOPSIES
60 .!
54%
I
40 -
30 20 10
I 1
I
..
01
OUANTITY OF HAEMOSIDERIN
=
ACUTE REJECTION
4b
TREATED REJECTION
BRONCHIOLAR HAEMOSIDERIN
% BIOPSIES
100
7
I
79%
.* QUANTITY OF HAEMOSIDERIN
=
ACUTE REJECTION
TREATED REJECTION
Fig. &Bar chart showing the extent of penvascular (a) and bronchiolar (b) haemosiderin during acute lung rejection and its extent after treatment
the next 12 months than did patients with normal therefore consistently demonstrated haemosiderin biopsies. Whether the same is true after an episode around vessels with or without a cellular infiltrate; of acute rejection remains uncertain. Further pro- however, after acute rejection the extent of the spective studies are necessary and are currently deposits increases. Haemosiderin is a useful marker of recent vascular injury and we have taken it to being undertaken. There was a tendency for haemosiderin deposits imply injury as a result of the perivascular infiltrate to be more extensive in the follow-up biopsies. It in ALR. This may have importance when considermust, however, be appreciated that many of the ing the chronic and irreversible consequences of rejection episodes studied were not the first episode acute rejection. A frequent non-specific finding in suffered by the patients. Haemosiderin was present TBBs of HLTs was the presence of numerous in biopsies as a result of a previous acute rejection alveolar haemosiderin-laden macrophages. We did episode and is removed very slowly. Some patients not include this feature in the analysis because it
LUNG HISTOLOGY AFTER TREATMENT OF REJECTION 5a
111
HISTOLOGICAL GRADE
% BIOPSIES
60
7
55%
1
50
0
1 2 GRADE OF REJECTION
ACUTE REJECTION
5b
3
TREATED REJECTION
FIBROSIS
% BIOPSIES
701
65%
I
43%
tt
.*
EXTENT OF FIBROSIS ACUTE REJECTION
TREATED REJECTION
Fig. %Bar charts to show the change in histological grade of ALR (a) (see Table I) and the degree of fibrosis determined by TBB before and after treatment (b)
was such a common finding in otherwise normal biopsies as well as in those of acute pulmonary infections and ALR. The bronchiolar infiltrates displayed many similarities to the perivascular infiltrates. However, there was less tendency for resolution of bronchiolar infiltrates after treatment. Explanations for the apparent resistance of inflammatory cells in this region to corticosteroids should perhaps be sought in the vascular hypothesis suggested for chronic lung rejection.16 Acute rejection affects both pulmonary and bronchial arteries and the intrapulmonary airways depend on collaterals from
pulmonary arteries retrogradely filling the bronchial arteries.” Reduced blood perfusion of the airway mucosa, seen experimentally in A L R , ’ ~ may therefore limit access of corticosteroid to the airway infiltrates. Topical treatment by inhaled corticosteroid aerosols may be a more appropriate approach for this problem.” We did not detect a significant increase in lung fibrosis after treatment, at least in the short term. The efficacy of TBB in detecting OB has been demonstrated by Yousem et al.’ and this has also been our experience. Submucosal fibrosis has been associated with the development of OB8but we have
112
C. A. CLELLAND ET AL.
found that as well as indicating past repeated lung rejection or infection, it can occur in patients who are well and have no severe airways obstruction.' Broncho-alveolar lavage (BAL) provides an opportunity to sample the cells involved in the rejection process in HLT. There are some similarities to TBB and this was the subject of an earlier report.20 Briefly, the absolute numbers and the percentage of lymphocytes increased in ALR, but there was much overlap with normal HLT and with infection. After augmented immunosuppression, lymphocytes and neutrophils diminish. Neutrophils are more numerous in the BAL of HLTs during rejection than would be expected from the histological appearances. We suspect that a significant number of neutrophils in the BAL are derived from airways larger than are usually sampled at TBB. Eosinophils are scarce in both BAL and TBB. In practical terms, our findings reinforce the need to review each biopsy in relation to previous biopsies and not in isolation. Close monitoring of patients, both clinically and by histology, should avoid the serious past problems of heart-lung transplantation.2' ACKNOWLEDGEMENTS
We are grateful for the editorial advice of Mr Ben Milstein. Thanks are due to Mrs A. Croker for typing the manuscript and to Mrs C. Richards for help with the photomicrographs. REFERENCES I . Hutter JA, Stewart S, Higenbottam TW, Wallwork J. Histological changes in heart-lung transplant recipients during rejection episodes and at routine biopsy. J Heurt Transplant 1988; 7: 440444. 2. Higenbottam TW, Penketh ARL, Stewart S, Wallwork J. Transbronchial lung biopsy to diagnose lung rejection and infection of heart-lung transplants. Transplanrarion 1988: 46: 532-539. 3. Yousem SA, Burke CM, Billingham ME. Pathologic pulmonary alterations in long-term human heart-lung transplantation. Hwn Pafhol'1985; 1 6 91 1-923.
4. Tazelaar HD, Yousem SA. The pathology of combined heart-lung transplantation: an autopsy study. Hum Parholl988;1 9 1403-1416. 5 . Clelland CA, Higenbottam TW,Otulana BA, er al. Histological
prognostic indicators for the lung allografts of heart-lung transplant recipients. J Heart Transplant (in press). 6. Cupps TR, Fauci AS. Corticosteroid-mediated immunoregulation in man. Immunol Rev 1982; 65: 133-155. 7 . Provoost AP. de Keyzer MH. Kort WJ, Wolf ED. Superiority of continuous infusion of prednisolone over daily injections in the prolongation of heart allograft survival in rats. Trunsplanrafion 1982; 34.221-222. 8. Scott J, Higenbottam TW, Hutter J, Clelland C, Wallwork J. The
natural history ofobliterative bronchiolitis in patients with heart-lung transplants. Trans Proc 1989; 21: 2592-2593. 9. Yousem SA, Paradis IL, Dauber JH, Griffith BP. Efficacy of transbronchial lung biopsy in the diagnosis of bronchiolitis obliterans in heart-lung transplant recipients. Transplanrarion 1989;47: 893-895. 10. Hakim M , Higenbottam TW. Bethune D, er a / .Selection and procurement of heart-lung grafts for transplantation. J Thorac Cardiovasc Surg 1988; 98:4 7 4 4 7 9 . 1 1 . Hutter JA, Despins D, Higenbottam TW, Stewart S, Wallwork J. Heart-lung transplantation: better use of resources. Am J Med 1988: 854-11. 12. Otulana BA, Higenbottam TW, Scott JP, Clelland C. Wallwork J.
Pulmonary function monitoring allows diagnosis of lung rejection in heart-lung transplant recipients. Transplanr Proe 1989; 21: 2583-2584. 13. Otulana BA, Higenbottam TW, Scott JP, Igboaka G, Wallwork J. The use of home spirometry in detecting acute lung rejection and infection following heart-lung transplantation. Chest (in press). 14. Millet B, Higenbottam TW, Flower C, Stewart S, Wallwork J. The radiographic appearances of infection and acute rejection of the lung following heart-lung transplantation. Am Rev Respir Dis 1989; 140: 62-67. 15. Penketh ARL, Higenbottam TW, Hutter J, Coutts C, Stewart S, Wallwork J. Clinical experience in the management of pulmonary opportunistic infection and rejection in heart-lung recipients. Thorux 1988; 4 3 762-169. 16. Yousem SA, Paradis IL, Dauber JH, er a / .Pulmonary arteriosclerosis in long-term human heart-lung transplant recipients. Transplantarion 1989; 47: 564-569. 17. Ladowski JS,Hardesty RL, Griffith BB. The pulmonary artery blood supply to the supracannal trachea. J Heart Transp/unr 1984;4: 40-42. 18. TanateH,YadaI,NamikawaS. KusagawaM. Earlydetectionoflung rejection by measurement of bronchial mucosal blood flow using laser doppler flowmeter. Trunsplanr Proc 1989; 21: 2590-2591. 19. Ruers TJM, Buurman WA, Smits JFM, er al. Local treatment of renal allografts, a promising way to reduce the dosage of immunosuppressive drugs. Transplanration 1986;41: 156-163. 20 Clelland CA, Higenbottam TW. Monk JA, er a / . A comparison of transbronchial lung biopsy appearances and cell profiles of bronchoalveolar lavage from heart-lung transplants. Am Rev Respir Dic (in press). 21 Scott JP, Higenbottam TW, Clelland C, ef al, The natural history of chronic rejection in heart-lung transplant recipients. J Heart Transplunr (in press).
161: 105-1 12 (1 990)
THE HISTOLOGICAL CHANGES IN TRANSBRONCHIAL BIOPSY AFTER TREATMENT OF ACUTE LUNG REJECTION IN HEART-LUNG TRANSPLANTS COLIN A. CLELLAND, TIMOTHY W. HIGENBOTTAM, SUSAN STEWART, JOHN P. SCOTT AND JOHN WALLWORK
The Heart-Lung Transplant Research Unit, Papworth Hospital, Papworth Everard, Cambridge CB3 8RE. U.K. Received 28 December 1989 Accepted 8 January 1990
SUMMARY Transbronchial lung biopsies taken during episodes of acute lung rejection in heart-lung transplant patients were examined histologically. When the diagnosis was confirmed by microscopy, the patients were treated with augmented immunosuppression by high-dose corticosteroids. A second biopsy was obtained an average of 23.5 days after commencement of treatment. These biopsies were examined to determine the histological changes caused by treatment. In most cases, there were both quantitative and morphological differences between the infiltrates in acute rejection and in the remaining perivascular infiltrates after treatment. In acute rejection, 26 of 28 biopsies contained perivascular lymphocytic infiltrates, lymphocytes being large and blast-like. Although 20 of 28 follow-up biopsies still contained perivascular infiltrates, they were smaller and the lymphocytes smaller in size. Half the biopsies in rejection contained neutrophils, but less than half contained eosinophils in the perivascular infiltrates. After treatment, all these cells were less numerous. Another feature of treated rejection was the presence of haemosiderin around vessels suggesting earlier acute vascular injury. However, haemosiderin persists long after the cellular infiltrate has disappeared and cannot be considered a reliable feature of recently treated acute lung rejection. The bronchiolar infiltrates showed a similar pattern of responses to the perivascular infiltrates. KEY woms-Heart-lung
transplantation, acute lung rejection, transbronchial lung biopsy, augmented immunosuppression.
INTRODUCTION T h e histopathology of acute lung rejection (ALR) in human heart-lung transplants (HLTs) has been described in studies o f transbronchial lung biopsies (TBBs) and post-mortem material.14 The use of the larger alligator forceps has provided good-sized TBBs, enabling the distinction to be made between ALR and pulmonary infection. Pyroninophilic, perivascular lymphocytic infiltrates are characteristic of ALR and are often associated with similar lymphocytic infiltration o f bronchioles which, in the absence o f infection, has also been attributed to ALR. Examination of TBBs, together with analysis of the clinical course of patients after HLT, has Addressee for correspondence: Dr T. W. Higenbottam, Department of Respiratory Physiology, Papworth Hospital, Papworth, Everard, Cambridge CB3 8RE,U.K.
0022-341 7/90/060101-08 $05.00 0 1990 by John Wiley & Sons, Ltd.
Table I-Histological classification of acute lung allograft rejection based on extent of inflammatory infiltration identified on TBB Grade
Histological appearances
0
No significant inflammation. Normal biopsy
1
Small, infrequent perivascular infiltrates with or without bronchiolar lymphocytic infiltrates
2
Larger, more frequent perivascular lymphocytic infiltrates with or without moderate bronchiolar lymphocytic inflammation. Occasional neutrophils and eosinophils
3
Extension of infiltrates into alveolar septa and alveolar spaces with or without bronchiolar mucosal ulceration
106
C. A. CLELLAND ET AL.
Table 11-Pre-operative diagnoses and mean ages of the 21 patients
Pre-operative diagnosis Eisenmenger’s syndrome Cystic fibrosis Pulmonary vascular disease Primary pulmonary hypertension Cryptogenic fibrosing alveolitis Sarcoidosis Churg-Strauss syndrome and pulmonary hypertension
No. of patients 8 7 1 2 1 1 1
Mean age in years
(SD) 29.6 (9.4) 22.7 (1-4) 22 23 (11) 53 43 34
Fig. 1-Transbronchial lung biopsies takenduringacutelung rejection (ALR)and aftertreatment in one patient. (a)In ALR, thereis a dense infiltrate of large lymphocytes around a small blood vessel. No neutrophils are seen. (b) After treatment, the penvascular infiltrate is smaller and the lymphocytesare reduced in size. Dark pigment can be seen even on haematoxylin and eosin staining
allowed a simple grading of histopathological severity of rejection to be devised (Table I) which helps to predict o ~ t c o r n e . ~ In the course of these studies, it has become clear that treatment, in particular augmented immunosuppression in the form of ‘pulsed’ steroids, modifies the histopathological appearance of ALR.
Corticosteroids are known to reduce lymphocytic as well as other cellular infiltrations of allo enic organ grafts both in man6 and in animals! Frequent episodes of acute rejection are also associated with an increased frequency of submucosal fibrosis and vascular sclerosis on TBB.’ Both histological features are also seen in those patients who have
LUNG HISTOLOGY AFTER TREATMENT OF REJECTION 2a
surgical technique, lung preservation system, and maintenance immunosuppressive treatment have been previously described in detail.”.” The patients were selected as each had experienced at least one episode of lung rejection. Methods of lung physi~logical”*’~ and chest radiological14 surveillance of these patients have been described earlier, together with clinical indications for TBB. The technique of TBB in HLT has been described,’ but in this study at least three biopsies were taken from each lobe of one lung (lingula being considered as a separate lobe). Processing and staining of the biopsies are now standard.’ Acute lung rejection in each patient was treated with 0-5-1 g methyl prednisolone intravenously daily for 3 days, and then oral prednisolone 1 mg/ kg/day in decreasing doses down to 10 mg daily over 10 days. Repeat TBBs were then performed, on average 23.5 days after diagnosis of ALR ( & 13,4s.D.; range: 7-58 days). Episodes of infection were excluded from the analysis. These were diagnosed by histology’ and by viral, bacterial, and fungal culture of TBB, concomitant bronchoalveolar lavage specimens, and sputa.
SIZE OF INFILTRATES
CELLS WIDE I 2 20 I I
i
ACUTE REJECTION
2b %
40
;
TREATED REJECTION
FREQUENCY
BIOPSIES 35%
107
35%
ANALYSIS
..
=
FREOUENCY OF INFILTRATES
ACUTE REJECTION
.*
TREATED REJECTION
Fig. 2-Size of perivascular infiltrates, expressed as number of cells wide (divided by 2), in ALR and TR (a) and the frequency of perivascular infiltrates around parenchymal vessels (-, none;
occasional; + +, several; and
+ + +, frequent infiltrates) (b)
The 28 pretreatment and 28 post-treatment TBBs were examined retrospectively by C.C., who had no knowledge of whether the biopsies were before or after treatment. The size and frequency of dense pyroninophilic lymphocytic perivascular infiltrates were assessed, Size was determined according to the
+, width of the infiltrate, measured by the number of
developed disabling or fatal obliterative bronchiolitis (OB).9 Here we report a study of 21 HLT patients who experienced 28 episodes of ALR. In each case, pulmonary infection was excluded by histopathology and culture. A comparison was made in each person between the histopathological appearance of TBB during ALR and TBB performed after ‘pulsed’ steroid therapy. PATIENTS AND METHODS Twenty-one of the 69 HLT patients from this institute were studied. They underwent surgery between July 1984 and July 1989. Their original diagnoses and ages are shown in Table 11. The
cells across the greatest diameter of the largest infiltrate of each biopsy. By convention, to give a measure of radius, this value was divided by two. The frequency of infiltrates was recorded semiquantitatively: none (-), occasional (+), frequent (+ +), or many (+ + +). Morphologically similar infiltration was sought in the bronchioles. The number of each type of inflammatory cell was also assessed semi-quantitatively using a scoring system of again none (-), occasional (+), or many (+ +). This scoring of cellularity was performed separately for infiltrates around parenchymal blood vessels and those around bronchioles. The presence of haemosiderin in these sites was similarly quantified. All the biopsies were allocated to a grade of severity using our recently reported classification system.’ Evidence of fibrosis was also recorded: (-) when there was none; (+) when
108
C. A. CLELLAND ETAL.
Table 111-Mean size of perivascular infiltrates,and numbers of different cell types in perivascular and bronchiolar infiltrates during ALR and after treatment. Estimates of haemosiderin deposition around parenchymal vessels and bronchioles are also included Acute rejection Mean SD
Treated rejection Mean SD
Perivascular infiltrates
Size of lymphocytic
7.1
(4.8)
infiltrate (cells wide -2) No. of lymphocytes (+’s) No. of plasma cells No. of neutrophils No. of eosinophils Amount of haemosiderin
1.32 0.11 0.61 0.50 0.61
(0.66) (0.31) (0.67) (0.63) (0.72)
1.18 0.29 0.39 0.32 0.25
(0.76) (0.45) (0.56) (0-47) (0.51)
3.8
(3.5)
0.75
(0.51) (0.31) (0.62) (0.54) (0.79)
0.11
0-39 0.18 0.86
Bronchiolar infiltrates (+ ’s)
No. of lymphocytes No. of plasma cells No. of neutrophils No. of eosinophils Amount of haemosiderin
0.93 0.29 0.32 0-11
0.61
(0.88) (0.52) (0.47) (0-41) (0.77)
the follow-up biopsies were free of perivascular lymphocytic infiltrates. There was no difference in the numbers of plasma cells before and after treatment and this cell type was only infrequently seen. There was a tendency for neutrophils to be reduced in numbers after treatment. A reduction in the small numbers of eosinophils present during ALR was RESULTS also seen after treatment. The cellular infiltrates The initial biopsies during ALR had infiltrates, affecting the bronchioles were similar on semiwhether perivascular or bronchiolar, of large blast- quantitative analysis to the perivascular infiltrates like lymphocytes with occasional neutrophils and (Table 111). There was less change in the response to eosinophils. After treatment, the lymphocytic infil- treatment than with perivascular infiltrates. Howtrates consisted mainly of small lymphocytes which ever, the trend of the changes in cell content was were more widely dispersed (Fig. 1). broadly similar. There was a slight increase in perivascular and Perivascular infiltrates were larger in ALR than in the paired follow-up biopsies in 21 of the 28 bronchiolar haemosiderin after treatment (Fig. 4). instances, the same size on four occasions, and As expected, because the basis of the histological smaller in three instances (Fig. 2a). The mean and classificationsystem rests on the intensity and extent standard deviation ( & SD) of the size of the infil- of lymphocytic infiltration of the biopsies the reductrates for the ALR and treated rejection (TR) tion in histological grade paralleled the changes groups are shown in Table 111. There was a reduc- found in the semi-quantitative analysis of perivastion in the frequency of infiltrates after treatment cular lymphocytes. The higher grades were affected (Fig. 2b). However, many follow-up biopsies still more by treatment than the lower grades (Fig. 5). contained either ‘occasional’or ‘several’ perivascu- The follow-up showed an increase in the number of lar infiltrates. Only 11 per cent of TBBs were free of biopsies with the mildest degree of fibrosis (intrainfiltrates during ALR as compared with nearly 30 alveolar and mild alveoIar septa1 fibrosis) (Fig. 5). per cent after treatment. No increase in severe fibrosis (submucosal bronAugmented immunosuppression diminished the chiolar fibrosis or obliterative airway fibrosis) was number of lymphocytes(Fig. 3). Nearly one-third of detected during this short-term period of follow-up. there was mild parenchymal fibrosis; (+ +) when parenchymal fibrosis was more extensive,and where there was bronchiolar submucosal fibrosis; and (+ + +) when there was biopsy evidence of fibrous obliteration of bronchioles.
109
LUNG HISTOLOGY AFTER TREATMENT OF REJECTION PLASMA CELLS
LYMPHOCYTES BIOPSIES
OF BIOPSIES
80
100
68%
70
60 60 40
30 20 10
..
..
0
OUANTITY OF PLASMA CELLS
QUANTITY OF LYMPHOCYTES ACUTE REJECTION
ACUTE REJECTION
TREATED REJECTION
FOSINOPHILS
NFlJTROPYlLS X BIOPSIES
X BIOPSIES 80
TREATED REJECTION
100
7
-
7%
QUANTITY OF NEUTROPHILS ACUTE REJECTION
..
..
TREATED REJECTION
7%
CIUANTITY OF EOSINOPHILS -ACUTE
REJECTION
TREATED REJECTION
Fig. %Bar charts to show assessment of the numbers of lymphocytes, plasma cells, neutrophils, and eosinophils in perivascular infiltrates in AR and TR (-, none; + ,few; + +, many)
these cell types were often reduced in numbers after treatment. One patient had persistent perivascular This paper extends our earlier studies on the infiltrates of eosinophils, but she had received HLT histology of ALR as seen on TBB.’**Paired TBBs for Churg-Strauss syndrome in association with were compared in each episode of ALR with those pulmonary hypertension. It is possible that this after completion of a course of augmented immuno- finding was related to recurrence of the primary suppressive treatment after rigorously excluding disease. On analysing the histological grade of ALR, it episodes of infection. This has enabled us to provide the first description of the histopathology of was apparent that many follow-up biopsies showed residual evidence of ALR in that small perivasular resolving rejection of the lung in man. Perivascular lymphocytic infiltrates were much infiltrates were still present. It can be questioned more frequent and larger in ALR than after treat- whether these patients should receive a further ment. Morphologically, the infiltrating lympho- course of ‘pulsed’steroids until the biopsies become cytes were smaller in size after treatment. Plasma clear of rejection. At resent, we cannot answer this. cells were infrequently observed, supporting the In an earlier study, patients who had predicted view that ALR is mainly a cell-mediated response. values for lung function and who had no respiratory Only in half the episodes of ALR were neutrophils symptoms or signs but had evidence of acute rejecof eosinophils found in significant numbers around tion on histology of TBB remained well despite no blood vessels. When present in the initial biopsy, treatment and had no more frequent rejection over
DISCUSSION
P
110
C. A. CLELLAND ET AL. 4a
PERIVASCULAR HAEMOSIDERIN
% BIOPSIES
60 .!
54%
I
40 -
30 20 10
I 1
I
..
01
OUANTITY OF HAEMOSIDERIN
=
ACUTE REJECTION
4b
TREATED REJECTION
BRONCHIOLAR HAEMOSIDERIN
% BIOPSIES
100
7
I
79%
.* QUANTITY OF HAEMOSIDERIN
=
ACUTE REJECTION
TREATED REJECTION
Fig. &Bar chart showing the extent of penvascular (a) and bronchiolar (b) haemosiderin during acute lung rejection and its extent after treatment
the next 12 months than did patients with normal therefore consistently demonstrated haemosiderin biopsies. Whether the same is true after an episode around vessels with or without a cellular infiltrate; of acute rejection remains uncertain. Further pro- however, after acute rejection the extent of the spective studies are necessary and are currently deposits increases. Haemosiderin is a useful marker of recent vascular injury and we have taken it to being undertaken. There was a tendency for haemosiderin deposits imply injury as a result of the perivascular infiltrate to be more extensive in the follow-up biopsies. It in ALR. This may have importance when considermust, however, be appreciated that many of the ing the chronic and irreversible consequences of rejection episodes studied were not the first episode acute rejection. A frequent non-specific finding in suffered by the patients. Haemosiderin was present TBBs of HLTs was the presence of numerous in biopsies as a result of a previous acute rejection alveolar haemosiderin-laden macrophages. We did episode and is removed very slowly. Some patients not include this feature in the analysis because it
LUNG HISTOLOGY AFTER TREATMENT OF REJECTION 5a
111
HISTOLOGICAL GRADE
% BIOPSIES
60
7
55%
1
50
0
1 2 GRADE OF REJECTION
ACUTE REJECTION
5b
3
TREATED REJECTION
FIBROSIS
% BIOPSIES
701
65%
I
43%
tt
.*
EXTENT OF FIBROSIS ACUTE REJECTION
TREATED REJECTION
Fig. %Bar charts to show the change in histological grade of ALR (a) (see Table I) and the degree of fibrosis determined by TBB before and after treatment (b)
was such a common finding in otherwise normal biopsies as well as in those of acute pulmonary infections and ALR. The bronchiolar infiltrates displayed many similarities to the perivascular infiltrates. However, there was less tendency for resolution of bronchiolar infiltrates after treatment. Explanations for the apparent resistance of inflammatory cells in this region to corticosteroids should perhaps be sought in the vascular hypothesis suggested for chronic lung rejection.16 Acute rejection affects both pulmonary and bronchial arteries and the intrapulmonary airways depend on collaterals from
pulmonary arteries retrogradely filling the bronchial arteries.” Reduced blood perfusion of the airway mucosa, seen experimentally in A L R , ’ ~ may therefore limit access of corticosteroid to the airway infiltrates. Topical treatment by inhaled corticosteroid aerosols may be a more appropriate approach for this problem.” We did not detect a significant increase in lung fibrosis after treatment, at least in the short term. The efficacy of TBB in detecting OB has been demonstrated by Yousem et al.’ and this has also been our experience. Submucosal fibrosis has been associated with the development of OB8but we have
112
C. A. CLELLAND ET AL.
found that as well as indicating past repeated lung rejection or infection, it can occur in patients who are well and have no severe airways obstruction.' Broncho-alveolar lavage (BAL) provides an opportunity to sample the cells involved in the rejection process in HLT. There are some similarities to TBB and this was the subject of an earlier report.20 Briefly, the absolute numbers and the percentage of lymphocytes increased in ALR, but there was much overlap with normal HLT and with infection. After augmented immunosuppression, lymphocytes and neutrophils diminish. Neutrophils are more numerous in the BAL of HLTs during rejection than would be expected from the histological appearances. We suspect that a significant number of neutrophils in the BAL are derived from airways larger than are usually sampled at TBB. Eosinophils are scarce in both BAL and TBB. In practical terms, our findings reinforce the need to review each biopsy in relation to previous biopsies and not in isolation. Close monitoring of patients, both clinically and by histology, should avoid the serious past problems of heart-lung transplantation.2' ACKNOWLEDGEMENTS
We are grateful for the editorial advice of Mr Ben Milstein. Thanks are due to Mrs A. Croker for typing the manuscript and to Mrs C. Richards for help with the photomicrographs. REFERENCES I . Hutter JA, Stewart S, Higenbottam TW, Wallwork J. Histological changes in heart-lung transplant recipients during rejection episodes and at routine biopsy. J Heurt Transplant 1988; 7: 440444. 2. Higenbottam TW, Penketh ARL, Stewart S, Wallwork J. Transbronchial lung biopsy to diagnose lung rejection and infection of heart-lung transplants. Transplanrarion 1988: 46: 532-539. 3. Yousem SA, Burke CM, Billingham ME. Pathologic pulmonary alterations in long-term human heart-lung transplantation. Hwn Pafhol'1985; 1 6 91 1-923.
4. Tazelaar HD, Yousem SA. The pathology of combined heart-lung transplantation: an autopsy study. Hum Parholl988;1 9 1403-1416. 5 . Clelland CA, Higenbottam TW,Otulana BA, er al. Histological
prognostic indicators for the lung allografts of heart-lung transplant recipients. J Heart Transplant (in press). 6. Cupps TR, Fauci AS. Corticosteroid-mediated immunoregulation in man. Immunol Rev 1982; 65: 133-155. 7 . Provoost AP. de Keyzer MH. Kort WJ, Wolf ED. Superiority of continuous infusion of prednisolone over daily injections in the prolongation of heart allograft survival in rats. Trunsplanrafion 1982; 34.221-222. 8. Scott J, Higenbottam TW, Hutter J, Clelland C, Wallwork J. The
natural history ofobliterative bronchiolitis in patients with heart-lung transplants. Trans Proc 1989; 21: 2592-2593. 9. Yousem SA, Paradis IL, Dauber JH, Griffith BP. Efficacy of transbronchial lung biopsy in the diagnosis of bronchiolitis obliterans in heart-lung transplant recipients. Transplanrarion 1989;47: 893-895. 10. Hakim M , Higenbottam TW. Bethune D, er a / .Selection and procurement of heart-lung grafts for transplantation. J Thorac Cardiovasc Surg 1988; 98:4 7 4 4 7 9 . 1 1 . Hutter JA, Despins D, Higenbottam TW, Stewart S, Wallwork J. Heart-lung transplantation: better use of resources. Am J Med 1988: 854-11. 12. Otulana BA, Higenbottam TW, Scott JP, Clelland C. Wallwork J.
Pulmonary function monitoring allows diagnosis of lung rejection in heart-lung transplant recipients. Transplanr Proe 1989; 21: 2583-2584. 13. Otulana BA, Higenbottam TW, Scott JP, Igboaka G, Wallwork J. The use of home spirometry in detecting acute lung rejection and infection following heart-lung transplantation. Chest (in press). 14. Millet B, Higenbottam TW, Flower C, Stewart S, Wallwork J. The radiographic appearances of infection and acute rejection of the lung following heart-lung transplantation. Am Rev Respir Dis 1989; 140: 62-67. 15. Penketh ARL, Higenbottam TW, Hutter J, Coutts C, Stewart S, Wallwork J. Clinical experience in the management of pulmonary opportunistic infection and rejection in heart-lung recipients. Thorux 1988; 4 3 762-169. 16. Yousem SA, Paradis IL, Dauber JH, er a / .Pulmonary arteriosclerosis in long-term human heart-lung transplant recipients. Transplantarion 1989; 47: 564-569. 17. Ladowski JS,Hardesty RL, Griffith BB. The pulmonary artery blood supply to the supracannal trachea. J Heart Transp/unr 1984;4: 40-42. 18. TanateH,YadaI,NamikawaS. KusagawaM. Earlydetectionoflung rejection by measurement of bronchial mucosal blood flow using laser doppler flowmeter. Trunsplanr Proc 1989; 21: 2590-2591. 19. Ruers TJM, Buurman WA, Smits JFM, er al. Local treatment of renal allografts, a promising way to reduce the dosage of immunosuppressive drugs. Transplanration 1986;41: 156-163. 20 Clelland CA, Higenbottam TW. Monk JA, er a / . A comparison of transbronchial lung biopsy appearances and cell profiles of bronchoalveolar lavage from heart-lung transplants. Am Rev Respir Dic (in press). 21 Scott JP, Higenbottam TW, Clelland C, ef al, The natural history of chronic rejection in heart-lung transplant recipients. J Heart Transplunr (in press).
