Clin. exp. Immunol. (1990) 81, 232-237
Pneumonitis in bone marrow transplant recipients results from a local immune response H. J. MILBURN*, R. M. DU BOIS*, H. G. PRENTICEt & L. W. POULTERt Departments of * Thoracic Medicine, t Haematology and t Immunology, Royal Free Hospital and School of Medicine, London, England (Acceptedfor publication 16 February 1990)
SUMMARY Eighteen recipients of allogeneic T cell-depleted bone marrow who developed 22 episodes of interstitial pneumonitis were investigated by bronchoalveolar lavage for the cause of pneumonitis. The cells obtained were examined using a panel of monoclonal antibodies with immunocytochemical techniques to identify lymphocyte subsets and the presence of surface molecules indicative of lymphocyte activation. The majority of patients had an excess of lymphocytes in lavage and most of these cells were positively stained with the McAb recognizing the CD8 antigen (suppressor/cytotoxic type T cells). Although the proportions of CD4+ (helper type) T cells were below normal, the absolute numbers were within normal limits, thus the CD4: CD8 ratio was consistently 1: 1 or less. A large proportion of the CD8+ cells displayed HLA-DR molecules (RFDRI+), interleukin-2 (IL-2) receptors (CD25+) and high concentration of CD7 antigen (RFT2+). Further analysis revealed that most CD8+ cells were CD5+ (RFTl +) yet a large proportion (20-40%) were CD5 -. A majority of CD8+ cells was also CD38+ (RFT10+) and Leu7+. No clear correlation between the emergence of a raised proportion of activated CD8 + cells and diagnosed cytomegalovirus infection was found. These results demonstrate, however, that cells with the phenotype of the resident T cells of the bronchial epithelium (CD8+CD5-) emerge to the air spaces and express activation markers. This raises the intriguing paradox of an aggressive local immune response occurring in an otherwise immunosuppressed group of patients. Keywords pneumonitis bone marrow transplantation bronchoalveolar lavage lymphocytes
INTRODUCTION As many as 50% of bone marrow transplant (BMT) recipients can develop interstitial pneumonitis in the six months immediately post-transplantation, and 70% of these (or 35% of all patients transplanted), may die from this complication (Neiman et al., 1977; Meyers, Fluornoy & Thomas, 1982; Buckner et al., 1984). Pneumonitis can be the result of infection by a variety of microorganisms, particularly cytomegalovirus (CMV) and Pneumocystis carinii, but frequently no infective cause is found. All BMT recipients have been rendered immunologically incompetent by their disease, the initial treatment and later by pretransplant conditioning which includes total body irradiation. In addition, some patients are treated with immunosuppressive drugs after transplant to prevent the development of
graft-versus-host disease (GVHD). Post-transplant immunosuppression has, however, been reduced in this centre since the donor marrow is now treated with monoclonal antibodies to deplete it of T cells, and GVHD has become uncommon Correspondence: Dr Heather Milburn, Department of Respiratory Medicine, Guy's Hospital, St Thomas Street, London SEI, England.
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(Prentice et al., 1984). For all these reasons it has been assumed that patients die from pneumonitis as a result of overwhelming infection which they are unable to counteract immunologically. As in other conditions, however, pneumonitis may be the result of an over-aggressive immune response in the lung, or even an initially appropriate immune response to a pathogen which, in the presence of systemic immunosuppression, fails to be regulated thus leading to local tissue destruction. In cryptogenic fibrosing alveolitis (CFA), for example, activation of both the humoral and cell-mediated responses in the lung have been demonstrated (Kravis et al., 1976; Hunninghake et al., 1981; Campbell et al., 1985). In addition, we have presented evidence for a humoral immune response in the lungs of BMT recipients with pneumonitis (Milburn et al., 1988) and have also reported an increase in both the total cell count and the absolute numbers of lymphocytes present in bronchoalveolar lavage (BAL) fluid from these patients suggesting a local immune associated inflammatory response despite systemic immunosuppression (Milburn et al., 1989). In the present study we have investigated further this population of alveolar lymphocytes for signs of activation. As the mucosal-associated immune system is widely distributed in
Pneumonitis in BMT recipients Table 1. Patient details
Patient no.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 episode 1 episode 2 16 episode 1 episode 2 17 episode 1 episode 2 18 episode 1 episode 2
Underlying disease
BAL diagnosis
CGL AML AML CGL CGL CGL ALL AML ALL CGL CGL CGL ALL AML AML
CMV CMV CMV
AML
CMV CMV+cryptosporidium CMV
AML ALL
CMV+P. carindi CMV+P. carindi P. carindi C. albicans C. albicans Ps. aeruginosa Measles Idiopathic Idiopathic Idiopathic Idiopathic CMV CMV
CMV Staph. aureus Staph. aureus
survival No Yes No No Yes No Yes Yes No No No Yes No Yes Yes No Yes No Yes Yes Yes No
CMV, Cytomegalovirus; CGL, chronic granulocytic leukaemia; AML, acute myeloid leukaemia; ALL, acute lymphoblastic leukaemia.
the lung and includes intra-epithelial lymphocytes as well as macrophage-like cells, we have used immunocytology with monoclonal antibodies (MoAbs), to determine whether the population of intra-epithelial lymphoctyes has decreased, as would be expected in a state of immunodeficiency, or increased, reflecting a state of immunoactivation. We have also looked for populations of lymphocytes which have previously been demonstrated in virus infections and GVHD in order to reveal the possible pathogenesis of the pneumonitis. SUBJECTS AND METHODS
Subjects Eighteen recipients of T cell-depleted allogeneic bone marrow were investigated. All were transplanted for haematological malignancies (Table 1) and all received conditioning with cyclophosphamide and total body irradiation with an average dose to the lungs of 670-830 cGy. Fourteen patients were men and four were women; their age range was 12-46 years (median 31 y). Only one patient smoked. A diagnosis of pneumonitis was made on the basis of symptoms (shortness of breath and fever) and a fall in the carbon monoxide gas transfer of > 20% predicted when compared with previous post-transplant measurements. In addition, the chest radiograph showed local changes in 12 cases, diffuse changes in nine, but was normal in one. With one exception (a patient who had been transferred from another hospital), all patients were investigated within 24 h of the onset of symptoms. The majority of patients developed pneumonitis between days 40 and 120 post-transplant, but three became ill more than a year after transplantation. Two ofthese latter patients had had
233
previous episodes of pneumonitis early after transplant, but these earlier episodes have not been included in this study. However, four patients developed two episodes of pneumonitis, both of which have been included in this study. Thus, a total of 22 episodes of pneumonitis were investigated. CMV was detected using both conventional cell culture and the detection of early antigen fluorescent foci (DEAFF) test (Griffiths et al., 1984) in BAL fluid from 11 of these patients. The diagnoses made in the remaining patients are outlined in Table 1. In addition, five healthy volunteers were bronchoscoped and lavaged to act as controls.
Sample collection BAL was performed using a BFTI fibreoptic bronchoscope. The upper airways were anaesthetized with 10% lignocaine topical spray, and 2 ml of 4% lignocaine were applied to the vocal cords under direct vision. Aliquots of 2% lignocaine were used to anaesthetize the lower respiratory tract. Supplemental oxygen was administered at a rate of 4 I/min throughout and for 4 h after the procedure. The tip of the bronchoscope was wedged into the orifice of a subsegmental bronchus of the middle lobe or lingula in patients with either normal chest radiographs or diffuse radiological changes. Where chest radiographs revealed local changes the appropriate segment was lavaged. BAL was performed with 3 x 60-ml aliquots of normal saline warmed to 370C and buffered to pH 7 4 by the addition of 175 pEq of sodium bicarbonate to 500 ml normal saline. Each aliquot was aspirated immediately after its instillation and collected in silicon-coated glass bottles maintained at 4°C. In addition, blood samples were taken from each patient at the time of bronchoscopy and the white cell count was determined. For ethical reasons, repeat bronchoscopies were not carried out unless patients developed a new episode of pneumonitis. Processing lavage samples All lavage samples were investigated for the presence of bacteria (including mycobacteria), fungi, protozoa and viruses by standard microscopy and culture. In addition, the total cell count was determined from an aliquot of untreated lavage fluid in a modified Neubauer haemocytometer, and viability was assessed by cellular exclusion of trypan blue ( > 70% in each case). Mucus strands were aspirated from the lavage fluid which was then centrifuged at 350 g for 10 min and the supernatant decanted. The cell pellet was washed twice in phosphate-buffered saline (PBS) (pH 7 2) and the cell suspension adjusted to a final concentration of 3 x 105 cells/ml. Aliquots of this suspension containing about 3 x 104 cells were used to make cytospin preparations in a Shandon Cytospin II (Shandon Instruments, Runcorn, UK). The cytospins obtained were air-dried for I h at room temperature, fixed in a chloroform/acetone mixture (1/ 1) for 10 min, air-dried again, wrapped in cling film and stored at - 20°C for future use. Differential cell counts were performed on cytospin preparations stained with May-GrunwaldGiemsa.
Immunocytochemistry Discrete subpopulations of lymphocytes were identified with monoclonal antibodies (MoAbs) by indirect immunocytochemical methods (Mason et al., 1983). The MoAbs used and the cell types identified are listed in Table 2. In addition, cytospin preparations were incubated with combinations of MoAbs
H. J. Milburn et al.
234
Table 2. Monoclonal antibodies used and cell types identified
Reagents used RFT mix (cocktail of RFT1, 8, 1 1) RFT2
RFTIO OKT4 RFT8 RFB mix
(cocktail of RFB6 & RFB4) Leu 7 Anti-Tac
Source
CD
Cells identified
CD5, 8,2
RFHSM
All T cells
CD7 CD38 CD4 CD8
RFHSM
RFHSM RFHSM
T blast/activated T cells Subset of T cells Helper type T cells Suppressor/cytotoxic type T cells All B cells
Beckton Dickinson Dr T. Uchiyama* RFHSM
NK cells and some T cells Interleukin-2 receptor Framework epitope HLA-DR
CD21/22 CD25
RFDR1
RFHSM
Ortho-pharmaceuticals
RFHSM, Royal Free Hospital School of Medicine. * NIH, Bethesda, MD. Table 3. CD4: CD8 (T4:T8) ratios in BAL fluid from BMT recipients with pneumonitis
Total T cells (mean% + s.d.)
Absolute numbers (cells x 105) CD4:CD8
All BMTpatients CMV+ CMVNormal range
CD4+
CD8+
CD4+
CD8+
(T4:T8)
11-5+9-5
57-1+27-6 61-9+24 51-8+26-8
0-15+0-01 0-1+0-01 0-2+0-02 004-006
0-5+0-14 0-25+0-06 0-8+0-2 0-01-0-03
1:3-3 (0-3)
13-3+8-2 10-3+9-0 50-80
15-40
followed by class-specific second layer antibodies conjugated to FITC or TRITC (indirect immunofluorescence) to assess double staining of lymphocytes. These preparations were examined using a Zeiss microscope with epi-illumination and selected barrier filters for FITC and TRITC. In each case, random high power fields from cytospins of each reaction were examined and the proportion of positive cells enumerated. Three-hundred cells were counted in each case. If no positive cells were detected, this was recorded at < 1%. The mean values of the proportion of cells in BAL fluids of each patient and the CD4/CD8 ratios were calculated. For the double-stained preparations, the percentages of CD8+ cells also expressing other markers were recorded. Appropriate positive tissue controls and reagent controls were used to ensure quality and reproducibility of the methods. Statistical analysis Statistical significance was determined by using Student's f-test for non-paired data. RESULTS Total cell count and differential The total cell count varied between patients with 14 lavages yielding considerably greater numbers of cells than would be expected from the normal lung (1-2 x 105 cells/ml). Numbers of cells as high as 20 times the normal range were recovered from some of these patients. The eight remaining lavages yielded total numbers of cells within the normal range.
1:2-5 (0-4) 1:4 (0-25) 2:1 (2)
Differential cell counts showed a significantly greater proportion and absolute number of lymphocytes than normal in most patients; 25 + 16 6% (mean + s.d.) of cells recovered were lymphocytes (normal range 8-12%). There were no consistent differences between patients diagnosed as CMV+ and others. These results have been described in detail elsewhere (Milburn et al., 1989). Subsets of lymphocytes There were high proportions (57 1 + 27-6% of total T cells) of CD8 + (suppressor/cytotoxic type) T cells and a 50-fold increase in the absolute number of these cells (0-5 + 014 x 105 cells/ml). Although the proportion of CD4+ (helper type) cells was reduced, the absolute numbers were slightly increased. The resulting ratios of CD4+ to CD8+ cells were consistently 1: 1 or lower in the transplant patients (Table 3). In normal lungs, a ratio of 2:1 is expected (Daniele et al., 1985). B cells were present in normal proportions (9-3+8-4% of lymphocytes) in lavage from most of the transplant patients, but absolute numbers were increased consistent with the increase in absolute numbers of lymphocytes (Fig. 1). Proportions of activated T cells The MoAb RFT2 (CD7) identifies an antigen (mol. wt 40 000) which, although present on all T cells, has been shown to be expressed more strongly on activated T cells (Poulter et al., 1985). Numbers of strongly CD7+ cells were counted and expressed as a percentage of total T cells. A significant proportion of T cells in patients with pneumonitis (mean 59-7%,
Pneumonitis in BMT recipients
235
100
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901-
0
90
01
01
80 [-
a
o4
00
80
0 0
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0
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8
a)
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0 *
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8 0
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A
0
ot
20
A~~
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01
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0
-
B Cells T Cells
CD4+ CD8+
A
CD7+
Fig. 1. Lymphocyte subsets in BAL from BMT patients with pneumonitis. *, CMV in BAL; 0, no CMV detected in BAL. Arrows are normal range.
range 10-96%; normal range 10-20%) strongly expressed the CD7 antigen, and significant numbers of T cells (28-7+ 16-8%) also expressed HLA-DR molecules (RFDR1 +). Combination immunofluorescence using MoAbs to CD8 antigens and activation markers revealed that 80% (mean) of CD8 + cells expressed CD7 antigens and 60% (mean) expressed CD25 antigens (interleukin-2 receptors) (Fig. 2). Although 25% (mean) of CD8+ lavage cells were HLA-DR+, this proportion was not above that found in normal lavage. Absolute numbers of HLA-DR+ CD8+ cells were, however, increased.
Phenotype of the CD8+ population Four further combinations of MoAbs were used to investigate the CD8+ T cell population. These were CD8/CD38, CD8/ CD5, CD8/Leu7 and CD38/Leu7. This analysis revealed that 70% of CD8+ cells were CD5+ (T1+); 50% were Leu7+ and 50% were CD38+ (T10+). Further analysis revealed that multiple phenotypically distinct subsets of CD8+ cells were These were in fluid. the present lavage CD8+CD5-Leu7-CD38+, the phenotype of activated intraepithelial lymphocytes (Selby, Janossy & Jewell, 1981); CD8+CD5+Leu7+CD38-, a phenotype consistent with the circulating cells found in virus infections (Carney et al., 1981; Crawford et al., 1981); CD8+CD5 -Leu7 -CD38-, a phenotype consistent with cells found in GVHD infiltrates of the skin (Favrot et al., 1983); CD8+CD5+Leu7+CD38+, the phenotype of circulating cells in BMT recipients with GVHD and/or overwhelming virus infection (Favrot et al., 1983); and
CD8+CD7 +
CD8+DR+
CD8+CD25+
Fig. 2. Activation markers on CD8 + cells in BAL: comparison between normals and BMT patients with pneumonitis. 0, CMV in BAL; 0, no CMV detected in BAL; *, normals.
CD8+CD5+Leu7-CD38+, a phenotype consistent with activated suppressor/cytotoxic type T cells. No single population was in a majority but CD38+CD5+Leu7+ constituted the largest population in percentage terms (see Discussion). Relation of cell type and number to time interval after transplant The immunocytochemical analysis of BAL fluid cells was related to the interval following transplantation to see whether the cells seen simply reflected the recovery of the bone marrow. Peripheral blood taken at the time of bronchoscopy contained a mean total white cell count of 3 5 + 1-6 x 109 cells/l (range 0- 118-5) in the transplant patients. Only six patients had normal total peripheral white cell counts (4-10 x 109 cells/l) and one had a raised count. The remainder had white cell counts below normal and none had a lymphocytosis. There was no relation between the peripheral blood differential count and the type of cells recovered in the lavage. Also, the proportion and numbers of CD8 + cells recovered from the lungs of patients with pneumonitis greatly exceeded the proportion and numbers of CD8+ cells in the peripheral blood. No relation was found between raised proportions of activated CD8+ cells in lavage fluid and the time after transplant when pneumonitis developed. DISCUSSION This study leads to two major conclusions. Firstly, the presence of a significantly raised population of CD8+ cells expressing CD7, CD25 and Leu7 antigens in the lavage of BMT patients
236
H. J. Milburn et al.
with pneumonitis suggests a local T cell-mediated immune response in a systemically immunosuppressed patient. Secondly, CD8+CD5- T cells are also present in the lavage fluid. This phenotype is characteristic of the population ofintraepithelial lymphocytes of mucus membranes (Selby et al., 1981). This shows that cells with the phenotype of normal resident epithelial lymphocytes are involved in the immune response and migrate through the epithelium into the air spaces. In patients receiving T cell depleted bone marrow, the CD8+ cells in the peripheral blood begin to regenerate after about day 40 post-transplant and reach near normal levels around day 70 (Janossy et al., 1986). This finding is in contrast with studies in which T cell depletion was not performed (Favrot et al., 1983), when absolute numbers of circulating CD8 + cells increased 2-4fold above normal levels from month 2 post-transplantation. As our patients all received T cell-depleted marrow, the greatly increased numbers of CD8 + cells seen in lavage cannot be explained merely as a reflection of the recovery of this population in the peripheral blood. In general, however, pneumonitis did not occur until the peripheral cells began to approach normal levels. The numbers of activated CD8+ cells in BAL fluid, however, greatly exceeded normal levels. This suggests that pneumonitis occurs when BMT recipients recover the ability to mount a cellular immune response. Recipients of BMT are conditioned with total body irradiation and both the method of delivery and the dose given may be critical to the subsequent development of pneumonitis, particularly when caused by CMV (Lowenthal, Jude & McMillan, 1981). Even minor radiation injury seems to predispose to reactivation of virus and subsequent inflammatory reaction in the lung. Severe viral infection, once established, may further activate suppressor/cytotoxic CD8+ cells and abolish helper effects, thus exacerbating the situation (Verdonck & de Gast, 1984). Radiation injury may also predispose to the development of a local graft-versus-host reaction which might be responsible for the high mortality seen with pulmonary infections in BMT patients. This is supported by experiments in the mouse which demonstrated that animals with GVHD infected with CMV develop a fatal infection, whereas CMV-infected, non-GVHD mice do not (Grundy et al., 1985). An excess of CD8+ cells has previously been demonstrated in the skin of patients with GVHD and a prolonged CD4+CD8+ imbalance in the peripheral blood of patients who receive non-T-depleted marrow (Favrot et al., 1983). However, these patients were far more likely to develop GVHD than our patients who all received Tdepleted transplants (Prentice et al., 1984). We have, however, demonstrated a population of CD8+ cells in the lung bearing the phenotype CD8+CD5-Leu7-CD38-. This population is phenotypically similar to the intra-epithelial lymphocytes in the gut (Selby et al., 1981) and in the bronchial mucosa (De Silva et al., personal communication) as well as to the cells which infiltrate the skin in GVHD (Favrot et al., 1983). Nevertheless, our patients had no obvious signs of GVHD. A further population of CD8+ cells in the lung in our patients was CD5+Leu7+CD38+HLA-DR+ and some of these cells also expressed the T cell activation antigen Tac (CD25+). These are similar to the circulating cells seen in BMT recipients who develop virus infections (Favrot et al., 1983) including CMV (Wursch et al., 1985). Circulating CD8+ cells of this kind are also found associated with infectious mononucleosis (Carney et al., 1981) or other infections (Reinherz, O'Brien &
T cells
CD8+ (T8+): Leu7 +
CD38+(RFT 10) :CD5 (RFTI) BMT Activated :Activaed: GVHD GVHD and/or virus: suppressor/ :local Iinfiltrates in skin (circulating cells) cytotoxic :cells cells :(circulating
Normals
1EBV and CMV
:cells)
Fig. 3. Schematic representation of subsets of CD8 + cells found in BAL of BMT recipients with pneumonitis. Horizontal lines represent the breadth of distribution, vertical columns denote phenotypically distinct subsets. Phenotype for each subset expressed by horizontal lines crossing vertical columns.
Rosenthal, 1980; Crawford et at., 198 1) in patients who have not been immunosuppressed. A similar subset has been observed in cardiac transplant recipients whose serology is consistent with active CMV infection-(Maher et at., 1985). These results suggest a CD8+ cell-mediated immune response to an infecting organism. As the number of these cells can be increased when the patient develops GVHD, the combination of such immunopathological sequelae may lead to further lung damage and subsequent morbidity and/or mortality. Using extensive 'double' analysis of BAL populations it is possible to construct a schematic diagram representing the relative distribution of surface antigens on CD8 + cells (Fig. 3). This approach may help to identify the character of the immune response underlying the pneumonitis, in that the majority of CD8+ T cells are seen to exhibit the same phenotype as those known to be involved in GVHD or found in the circulation of patients with viral infection. However, as it is known that these phenotypes can occur in other conditions, this proposal must remain conjecture. The high incidence of opportunistic infections seen in BMT patients has led to the general assumption that subsequent pneumonitis and death is a direct result of an immunocompromised patient being unable to combat an overwhelming infection. This theory. however, is not supported by the major observation of this paper, which is the incidence in BAL of increased numbers of CD8+CD38+Leu7+CD25+ cells, a phenotype characteristic of activated T cells involved in an immune response against infection. In many cases anti-microbial or antiviral treatment is effective against the organism responsible but does not improve the outcome of pneumonitis (Shepp et at., 1985). It seems unlikely that death from pneumonitis is simply due to damage caused by infection. Moreover, it has been shown that reducing viral growth in mice moderated but did not prevent CMV pneumonitis (Shanley & Pesanti, 1985). Taken together, these observations suggest that the pneumonitis results predominantly from an aggressive immune response. The presence of admixed CD8+CD5- T cells within the lavage adds weight to the suggestion that this response is a local phenomenon, as such T cells have been shown to be the resident population of the bronchial epithelium and rare among circulat-
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Hydroxy-l-(hydroxymethyl) ethoxymethyl) guanine in the treatment of cytomegalovirus pneumonia. Ann. intern. Med. 103, 368. VERDONCK, L.F. & DE GAST, G.C. (1984) Is CMV infection a major cause of T cell alterations after (autologous) BMT? Lancet, i, 932. WURSCH, A.M., GRATAMA, J.W., MIDDLELDORP, J.M., NISSEN, C., GRATWOHL, A., SPECK, B., JANSEN, J., D'AMARO, J., THE, T.H. & DE GAST G.C. (1985) The effect of cytomegalovirus on T lymphocytes after allogeneic bone marrow transplantation. Clin. exp. Immunol. 62, 278.
Pneumonitis in bone marrow transplant recipients results from a local immune response H. J. MILBURN*, R. M. DU BOIS*, H. G. PRENTICEt & L. W. POULTERt Departments of * Thoracic Medicine, t Haematology and t Immunology, Royal Free Hospital and School of Medicine, London, England (Acceptedfor publication 16 February 1990)
SUMMARY Eighteen recipients of allogeneic T cell-depleted bone marrow who developed 22 episodes of interstitial pneumonitis were investigated by bronchoalveolar lavage for the cause of pneumonitis. The cells obtained were examined using a panel of monoclonal antibodies with immunocytochemical techniques to identify lymphocyte subsets and the presence of surface molecules indicative of lymphocyte activation. The majority of patients had an excess of lymphocytes in lavage and most of these cells were positively stained with the McAb recognizing the CD8 antigen (suppressor/cytotoxic type T cells). Although the proportions of CD4+ (helper type) T cells were below normal, the absolute numbers were within normal limits, thus the CD4: CD8 ratio was consistently 1: 1 or less. A large proportion of the CD8+ cells displayed HLA-DR molecules (RFDRI+), interleukin-2 (IL-2) receptors (CD25+) and high concentration of CD7 antigen (RFT2+). Further analysis revealed that most CD8+ cells were CD5+ (RFTl +) yet a large proportion (20-40%) were CD5 -. A majority of CD8+ cells was also CD38+ (RFT10+) and Leu7+. No clear correlation between the emergence of a raised proportion of activated CD8 + cells and diagnosed cytomegalovirus infection was found. These results demonstrate, however, that cells with the phenotype of the resident T cells of the bronchial epithelium (CD8+CD5-) emerge to the air spaces and express activation markers. This raises the intriguing paradox of an aggressive local immune response occurring in an otherwise immunosuppressed group of patients. Keywords pneumonitis bone marrow transplantation bronchoalveolar lavage lymphocytes
INTRODUCTION As many as 50% of bone marrow transplant (BMT) recipients can develop interstitial pneumonitis in the six months immediately post-transplantation, and 70% of these (or 35% of all patients transplanted), may die from this complication (Neiman et al., 1977; Meyers, Fluornoy & Thomas, 1982; Buckner et al., 1984). Pneumonitis can be the result of infection by a variety of microorganisms, particularly cytomegalovirus (CMV) and Pneumocystis carinii, but frequently no infective cause is found. All BMT recipients have been rendered immunologically incompetent by their disease, the initial treatment and later by pretransplant conditioning which includes total body irradiation. In addition, some patients are treated with immunosuppressive drugs after transplant to prevent the development of
graft-versus-host disease (GVHD). Post-transplant immunosuppression has, however, been reduced in this centre since the donor marrow is now treated with monoclonal antibodies to deplete it of T cells, and GVHD has become uncommon Correspondence: Dr Heather Milburn, Department of Respiratory Medicine, Guy's Hospital, St Thomas Street, London SEI, England.
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(Prentice et al., 1984). For all these reasons it has been assumed that patients die from pneumonitis as a result of overwhelming infection which they are unable to counteract immunologically. As in other conditions, however, pneumonitis may be the result of an over-aggressive immune response in the lung, or even an initially appropriate immune response to a pathogen which, in the presence of systemic immunosuppression, fails to be regulated thus leading to local tissue destruction. In cryptogenic fibrosing alveolitis (CFA), for example, activation of both the humoral and cell-mediated responses in the lung have been demonstrated (Kravis et al., 1976; Hunninghake et al., 1981; Campbell et al., 1985). In addition, we have presented evidence for a humoral immune response in the lungs of BMT recipients with pneumonitis (Milburn et al., 1988) and have also reported an increase in both the total cell count and the absolute numbers of lymphocytes present in bronchoalveolar lavage (BAL) fluid from these patients suggesting a local immune associated inflammatory response despite systemic immunosuppression (Milburn et al., 1989). In the present study we have investigated further this population of alveolar lymphocytes for signs of activation. As the mucosal-associated immune system is widely distributed in
Pneumonitis in BMT recipients Table 1. Patient details
Patient no.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 episode 1 episode 2 16 episode 1 episode 2 17 episode 1 episode 2 18 episode 1 episode 2
Underlying disease
BAL diagnosis
CGL AML AML CGL CGL CGL ALL AML ALL CGL CGL CGL ALL AML AML
CMV CMV CMV
AML
CMV CMV+cryptosporidium CMV
AML ALL
CMV+P. carindi CMV+P. carindi P. carindi C. albicans C. albicans Ps. aeruginosa Measles Idiopathic Idiopathic Idiopathic Idiopathic CMV CMV
CMV Staph. aureus Staph. aureus
survival No Yes No No Yes No Yes Yes No No No Yes No Yes Yes No Yes No Yes Yes Yes No
CMV, Cytomegalovirus; CGL, chronic granulocytic leukaemia; AML, acute myeloid leukaemia; ALL, acute lymphoblastic leukaemia.
the lung and includes intra-epithelial lymphocytes as well as macrophage-like cells, we have used immunocytology with monoclonal antibodies (MoAbs), to determine whether the population of intra-epithelial lymphoctyes has decreased, as would be expected in a state of immunodeficiency, or increased, reflecting a state of immunoactivation. We have also looked for populations of lymphocytes which have previously been demonstrated in virus infections and GVHD in order to reveal the possible pathogenesis of the pneumonitis. SUBJECTS AND METHODS
Subjects Eighteen recipients of T cell-depleted allogeneic bone marrow were investigated. All were transplanted for haematological malignancies (Table 1) and all received conditioning with cyclophosphamide and total body irradiation with an average dose to the lungs of 670-830 cGy. Fourteen patients were men and four were women; their age range was 12-46 years (median 31 y). Only one patient smoked. A diagnosis of pneumonitis was made on the basis of symptoms (shortness of breath and fever) and a fall in the carbon monoxide gas transfer of > 20% predicted when compared with previous post-transplant measurements. In addition, the chest radiograph showed local changes in 12 cases, diffuse changes in nine, but was normal in one. With one exception (a patient who had been transferred from another hospital), all patients were investigated within 24 h of the onset of symptoms. The majority of patients developed pneumonitis between days 40 and 120 post-transplant, but three became ill more than a year after transplantation. Two ofthese latter patients had had
233
previous episodes of pneumonitis early after transplant, but these earlier episodes have not been included in this study. However, four patients developed two episodes of pneumonitis, both of which have been included in this study. Thus, a total of 22 episodes of pneumonitis were investigated. CMV was detected using both conventional cell culture and the detection of early antigen fluorescent foci (DEAFF) test (Griffiths et al., 1984) in BAL fluid from 11 of these patients. The diagnoses made in the remaining patients are outlined in Table 1. In addition, five healthy volunteers were bronchoscoped and lavaged to act as controls.
Sample collection BAL was performed using a BFTI fibreoptic bronchoscope. The upper airways were anaesthetized with 10% lignocaine topical spray, and 2 ml of 4% lignocaine were applied to the vocal cords under direct vision. Aliquots of 2% lignocaine were used to anaesthetize the lower respiratory tract. Supplemental oxygen was administered at a rate of 4 I/min throughout and for 4 h after the procedure. The tip of the bronchoscope was wedged into the orifice of a subsegmental bronchus of the middle lobe or lingula in patients with either normal chest radiographs or diffuse radiological changes. Where chest radiographs revealed local changes the appropriate segment was lavaged. BAL was performed with 3 x 60-ml aliquots of normal saline warmed to 370C and buffered to pH 7 4 by the addition of 175 pEq of sodium bicarbonate to 500 ml normal saline. Each aliquot was aspirated immediately after its instillation and collected in silicon-coated glass bottles maintained at 4°C. In addition, blood samples were taken from each patient at the time of bronchoscopy and the white cell count was determined. For ethical reasons, repeat bronchoscopies were not carried out unless patients developed a new episode of pneumonitis. Processing lavage samples All lavage samples were investigated for the presence of bacteria (including mycobacteria), fungi, protozoa and viruses by standard microscopy and culture. In addition, the total cell count was determined from an aliquot of untreated lavage fluid in a modified Neubauer haemocytometer, and viability was assessed by cellular exclusion of trypan blue ( > 70% in each case). Mucus strands were aspirated from the lavage fluid which was then centrifuged at 350 g for 10 min and the supernatant decanted. The cell pellet was washed twice in phosphate-buffered saline (PBS) (pH 7 2) and the cell suspension adjusted to a final concentration of 3 x 105 cells/ml. Aliquots of this suspension containing about 3 x 104 cells were used to make cytospin preparations in a Shandon Cytospin II (Shandon Instruments, Runcorn, UK). The cytospins obtained were air-dried for I h at room temperature, fixed in a chloroform/acetone mixture (1/ 1) for 10 min, air-dried again, wrapped in cling film and stored at - 20°C for future use. Differential cell counts were performed on cytospin preparations stained with May-GrunwaldGiemsa.
Immunocytochemistry Discrete subpopulations of lymphocytes were identified with monoclonal antibodies (MoAbs) by indirect immunocytochemical methods (Mason et al., 1983). The MoAbs used and the cell types identified are listed in Table 2. In addition, cytospin preparations were incubated with combinations of MoAbs
H. J. Milburn et al.
234
Table 2. Monoclonal antibodies used and cell types identified
Reagents used RFT mix (cocktail of RFT1, 8, 1 1) RFT2
RFTIO OKT4 RFT8 RFB mix
(cocktail of RFB6 & RFB4) Leu 7 Anti-Tac
Source
CD
Cells identified
CD5, 8,2
RFHSM
All T cells
CD7 CD38 CD4 CD8
RFHSM
RFHSM RFHSM
T blast/activated T cells Subset of T cells Helper type T cells Suppressor/cytotoxic type T cells All B cells
Beckton Dickinson Dr T. Uchiyama* RFHSM
NK cells and some T cells Interleukin-2 receptor Framework epitope HLA-DR
CD21/22 CD25
RFDR1
RFHSM
Ortho-pharmaceuticals
RFHSM, Royal Free Hospital School of Medicine. * NIH, Bethesda, MD. Table 3. CD4: CD8 (T4:T8) ratios in BAL fluid from BMT recipients with pneumonitis
Total T cells (mean% + s.d.)
Absolute numbers (cells x 105) CD4:CD8
All BMTpatients CMV+ CMVNormal range
CD4+
CD8+
CD4+
CD8+
(T4:T8)
11-5+9-5
57-1+27-6 61-9+24 51-8+26-8
0-15+0-01 0-1+0-01 0-2+0-02 004-006
0-5+0-14 0-25+0-06 0-8+0-2 0-01-0-03
1:3-3 (0-3)
13-3+8-2 10-3+9-0 50-80
15-40
followed by class-specific second layer antibodies conjugated to FITC or TRITC (indirect immunofluorescence) to assess double staining of lymphocytes. These preparations were examined using a Zeiss microscope with epi-illumination and selected barrier filters for FITC and TRITC. In each case, random high power fields from cytospins of each reaction were examined and the proportion of positive cells enumerated. Three-hundred cells were counted in each case. If no positive cells were detected, this was recorded at < 1%. The mean values of the proportion of cells in BAL fluids of each patient and the CD4/CD8 ratios were calculated. For the double-stained preparations, the percentages of CD8+ cells also expressing other markers were recorded. Appropriate positive tissue controls and reagent controls were used to ensure quality and reproducibility of the methods. Statistical analysis Statistical significance was determined by using Student's f-test for non-paired data. RESULTS Total cell count and differential The total cell count varied between patients with 14 lavages yielding considerably greater numbers of cells than would be expected from the normal lung (1-2 x 105 cells/ml). Numbers of cells as high as 20 times the normal range were recovered from some of these patients. The eight remaining lavages yielded total numbers of cells within the normal range.
1:2-5 (0-4) 1:4 (0-25) 2:1 (2)
Differential cell counts showed a significantly greater proportion and absolute number of lymphocytes than normal in most patients; 25 + 16 6% (mean + s.d.) of cells recovered were lymphocytes (normal range 8-12%). There were no consistent differences between patients diagnosed as CMV+ and others. These results have been described in detail elsewhere (Milburn et al., 1989). Subsets of lymphocytes There were high proportions (57 1 + 27-6% of total T cells) of CD8 + (suppressor/cytotoxic type) T cells and a 50-fold increase in the absolute number of these cells (0-5 + 014 x 105 cells/ml). Although the proportion of CD4+ (helper type) cells was reduced, the absolute numbers were slightly increased. The resulting ratios of CD4+ to CD8+ cells were consistently 1: 1 or lower in the transplant patients (Table 3). In normal lungs, a ratio of 2:1 is expected (Daniele et al., 1985). B cells were present in normal proportions (9-3+8-4% of lymphocytes) in lavage from most of the transplant patients, but absolute numbers were increased consistent with the increase in absolute numbers of lymphocytes (Fig. 1). Proportions of activated T cells The MoAb RFT2 (CD7) identifies an antigen (mol. wt 40 000) which, although present on all T cells, has been shown to be expressed more strongly on activated T cells (Poulter et al., 1985). Numbers of strongly CD7+ cells were counted and expressed as a percentage of total T cells. A significant proportion of T cells in patients with pneumonitis (mean 59-7%,
Pneumonitis in BMT recipients
235
100
&O
901-
0
90
01
01
80 [-
a
o4
00
80
0 0
70
0
_
0
0
0
70
0 0
0
601-
01
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-
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(
8
a)
C.)
0
50
0 *
F
0
8 0
0 0
I
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E
40
60
0)
C)
0
-
o 0-
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S4 I-.
.
0
0
30 _
401
A
A
-0
08
0
-
o
0
30
0
A A
0
20 _
A
0
0 0
0
A 0
0
00
10
A
0
ot
20
A~~
A
01
A A
0
10
0
-
B Cells T Cells
CD4+ CD8+
A
CD7+
Fig. 1. Lymphocyte subsets in BAL from BMT patients with pneumonitis. *, CMV in BAL; 0, no CMV detected in BAL. Arrows are normal range.
range 10-96%; normal range 10-20%) strongly expressed the CD7 antigen, and significant numbers of T cells (28-7+ 16-8%) also expressed HLA-DR molecules (RFDR1 +). Combination immunofluorescence using MoAbs to CD8 antigens and activation markers revealed that 80% (mean) of CD8 + cells expressed CD7 antigens and 60% (mean) expressed CD25 antigens (interleukin-2 receptors) (Fig. 2). Although 25% (mean) of CD8+ lavage cells were HLA-DR+, this proportion was not above that found in normal lavage. Absolute numbers of HLA-DR+ CD8+ cells were, however, increased.
Phenotype of the CD8+ population Four further combinations of MoAbs were used to investigate the CD8+ T cell population. These were CD8/CD38, CD8/ CD5, CD8/Leu7 and CD38/Leu7. This analysis revealed that 70% of CD8+ cells were CD5+ (T1+); 50% were Leu7+ and 50% were CD38+ (T10+). Further analysis revealed that multiple phenotypically distinct subsets of CD8+ cells were These were in fluid. the present lavage CD8+CD5-Leu7-CD38+, the phenotype of activated intraepithelial lymphocytes (Selby, Janossy & Jewell, 1981); CD8+CD5+Leu7+CD38-, a phenotype consistent with the circulating cells found in virus infections (Carney et al., 1981; Crawford et al., 1981); CD8+CD5 -Leu7 -CD38-, a phenotype consistent with cells found in GVHD infiltrates of the skin (Favrot et al., 1983); CD8+CD5+Leu7+CD38+, the phenotype of circulating cells in BMT recipients with GVHD and/or overwhelming virus infection (Favrot et al., 1983); and
CD8+CD7 +
CD8+DR+
CD8+CD25+
Fig. 2. Activation markers on CD8 + cells in BAL: comparison between normals and BMT patients with pneumonitis. 0, CMV in BAL; 0, no CMV detected in BAL; *, normals.
CD8+CD5+Leu7-CD38+, a phenotype consistent with activated suppressor/cytotoxic type T cells. No single population was in a majority but CD38+CD5+Leu7+ constituted the largest population in percentage terms (see Discussion). Relation of cell type and number to time interval after transplant The immunocytochemical analysis of BAL fluid cells was related to the interval following transplantation to see whether the cells seen simply reflected the recovery of the bone marrow. Peripheral blood taken at the time of bronchoscopy contained a mean total white cell count of 3 5 + 1-6 x 109 cells/l (range 0- 118-5) in the transplant patients. Only six patients had normal total peripheral white cell counts (4-10 x 109 cells/l) and one had a raised count. The remainder had white cell counts below normal and none had a lymphocytosis. There was no relation between the peripheral blood differential count and the type of cells recovered in the lavage. Also, the proportion and numbers of CD8 + cells recovered from the lungs of patients with pneumonitis greatly exceeded the proportion and numbers of CD8+ cells in the peripheral blood. No relation was found between raised proportions of activated CD8+ cells in lavage fluid and the time after transplant when pneumonitis developed. DISCUSSION This study leads to two major conclusions. Firstly, the presence of a significantly raised population of CD8+ cells expressing CD7, CD25 and Leu7 antigens in the lavage of BMT patients
236
H. J. Milburn et al.
with pneumonitis suggests a local T cell-mediated immune response in a systemically immunosuppressed patient. Secondly, CD8+CD5- T cells are also present in the lavage fluid. This phenotype is characteristic of the population ofintraepithelial lymphocytes of mucus membranes (Selby et al., 1981). This shows that cells with the phenotype of normal resident epithelial lymphocytes are involved in the immune response and migrate through the epithelium into the air spaces. In patients receiving T cell depleted bone marrow, the CD8+ cells in the peripheral blood begin to regenerate after about day 40 post-transplant and reach near normal levels around day 70 (Janossy et al., 1986). This finding is in contrast with studies in which T cell depletion was not performed (Favrot et al., 1983), when absolute numbers of circulating CD8 + cells increased 2-4fold above normal levels from month 2 post-transplantation. As our patients all received T cell-depleted marrow, the greatly increased numbers of CD8 + cells seen in lavage cannot be explained merely as a reflection of the recovery of this population in the peripheral blood. In general, however, pneumonitis did not occur until the peripheral cells began to approach normal levels. The numbers of activated CD8+ cells in BAL fluid, however, greatly exceeded normal levels. This suggests that pneumonitis occurs when BMT recipients recover the ability to mount a cellular immune response. Recipients of BMT are conditioned with total body irradiation and both the method of delivery and the dose given may be critical to the subsequent development of pneumonitis, particularly when caused by CMV (Lowenthal, Jude & McMillan, 1981). Even minor radiation injury seems to predispose to reactivation of virus and subsequent inflammatory reaction in the lung. Severe viral infection, once established, may further activate suppressor/cytotoxic CD8+ cells and abolish helper effects, thus exacerbating the situation (Verdonck & de Gast, 1984). Radiation injury may also predispose to the development of a local graft-versus-host reaction which might be responsible for the high mortality seen with pulmonary infections in BMT patients. This is supported by experiments in the mouse which demonstrated that animals with GVHD infected with CMV develop a fatal infection, whereas CMV-infected, non-GVHD mice do not (Grundy et al., 1985). An excess of CD8+ cells has previously been demonstrated in the skin of patients with GVHD and a prolonged CD4+CD8+ imbalance in the peripheral blood of patients who receive non-T-depleted marrow (Favrot et al., 1983). However, these patients were far more likely to develop GVHD than our patients who all received Tdepleted transplants (Prentice et al., 1984). We have, however, demonstrated a population of CD8+ cells in the lung bearing the phenotype CD8+CD5-Leu7-CD38-. This population is phenotypically similar to the intra-epithelial lymphocytes in the gut (Selby et al., 1981) and in the bronchial mucosa (De Silva et al., personal communication) as well as to the cells which infiltrate the skin in GVHD (Favrot et al., 1983). Nevertheless, our patients had no obvious signs of GVHD. A further population of CD8+ cells in the lung in our patients was CD5+Leu7+CD38+HLA-DR+ and some of these cells also expressed the T cell activation antigen Tac (CD25+). These are similar to the circulating cells seen in BMT recipients who develop virus infections (Favrot et al., 1983) including CMV (Wursch et al., 1985). Circulating CD8+ cells of this kind are also found associated with infectious mononucleosis (Carney et al., 1981) or other infections (Reinherz, O'Brien &
T cells
CD8+ (T8+): Leu7 +
CD38+(RFT 10) :CD5 (RFTI) BMT Activated :Activaed: GVHD GVHD and/or virus: suppressor/ :local Iinfiltrates in skin (circulating cells) cytotoxic :cells cells :(circulating
Normals
1EBV and CMV
:cells)
Fig. 3. Schematic representation of subsets of CD8 + cells found in BAL of BMT recipients with pneumonitis. Horizontal lines represent the breadth of distribution, vertical columns denote phenotypically distinct subsets. Phenotype for each subset expressed by horizontal lines crossing vertical columns.
Rosenthal, 1980; Crawford et at., 198 1) in patients who have not been immunosuppressed. A similar subset has been observed in cardiac transplant recipients whose serology is consistent with active CMV infection-(Maher et at., 1985). These results suggest a CD8+ cell-mediated immune response to an infecting organism. As the number of these cells can be increased when the patient develops GVHD, the combination of such immunopathological sequelae may lead to further lung damage and subsequent morbidity and/or mortality. Using extensive 'double' analysis of BAL populations it is possible to construct a schematic diagram representing the relative distribution of surface antigens on CD8 + cells (Fig. 3). This approach may help to identify the character of the immune response underlying the pneumonitis, in that the majority of CD8+ T cells are seen to exhibit the same phenotype as those known to be involved in GVHD or found in the circulation of patients with viral infection. However, as it is known that these phenotypes can occur in other conditions, this proposal must remain conjecture. The high incidence of opportunistic infections seen in BMT patients has led to the general assumption that subsequent pneumonitis and death is a direct result of an immunocompromised patient being unable to combat an overwhelming infection. This theory. however, is not supported by the major observation of this paper, which is the incidence in BAL of increased numbers of CD8+CD38+Leu7+CD25+ cells, a phenotype characteristic of activated T cells involved in an immune response against infection. In many cases anti-microbial or antiviral treatment is effective against the organism responsible but does not improve the outcome of pneumonitis (Shepp et at., 1985). It seems unlikely that death from pneumonitis is simply due to damage caused by infection. Moreover, it has been shown that reducing viral growth in mice moderated but did not prevent CMV pneumonitis (Shanley & Pesanti, 1985). Taken together, these observations suggest that the pneumonitis results predominantly from an aggressive immune response. The presence of admixed CD8+CD5- T cells within the lavage adds weight to the suggestion that this response is a local phenomenon, as such T cells have been shown to be the resident population of the bronchial epithelium and rare among circulat-
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