Alveolar Macrophage Migration after Lung 'lransplantatlcn'?
ROBERT M. HOFFMAN, JAMES H. DAUBER, IRVIN L. PARADIS, BARTLEY P. GRIFFITH, and ROBERT L. HARDESTY
Introduction
Pulmonary infection has been a major source of morbidity and mortality in all immunosuppressed organ transplant recipients, but in recipients of a lung allograft it is particularly troublesome (1-3). The factors responsible for the higher than expectedfrequency and mortality are unclear at present, but defective pulmonary host defense mechanisms of the lung allograft probably playa key role. The alveolar macrophage (AM) is the resident phagocytic cell in the lung, and it is responsible for maintaining sterility in the distal airspaces. However, its function may be abnormal in the transplanted lung not only because of the effects of immunosuppressive drugs and infection, but also because it exists in a potentially hostile allogeneic environment (4). In this study AM were recovered from bronchoalveolar lavage performed on lung transplant recipients and assayedfor migratory activity. We elected to study this particular function because it is thought to be important in the early response to the deposition of bacteria in the distal airspaces of the lung. Wecompared AM migration between normal individuals and lung allograft recipients who weresubclassified according to their clinical status. In addition, the migratory activity of AM was tracked in a sequential fashion so that variations in migration within an individual patient could be correlated with that patient's clinical status. Methods Lung Transplant Recipients Lung transplant recipients were followed prospectively between July 1984 and May 1989. Of the 28 patients in the study 26 received lung and heart allografts for primary or secondary pulmonary hypertension refractory to medical therapy. Patients with secondary hypertension had intracardiac shunts or, in one case, pulmonary eosinophilic granuloma (5). Because the emphasis of this report is on the lung allograft, these patients are referred to as lung transplant recipients. 1\vo 834
SUMMARY Pulmonary Infection Is a major source of morbidity and mortality In recipients of lung allografts. The alveolar macrophage plays an Important role In pulmonary host defense, and to fulfill this role It must have the ability to orient and migrate In the direction of a stimulus. Thus migratory activity was measured In cells recovered from lung transplant recipients by bronchoalveolar lavage. The primary patient group consisted of recipients who had no evidence of Infection or rejection at the time of bronchoalveolar lavage. These patients were further subdlvldad Into an early postoperative group (less than 6 wk posttransplant) and a late postoperative group (greater than 6 wk posttransplant). Other categories Included patients with chronic rejection and a small group of patients with Pneumocystls car/nll pneumonia. Alveolar macrophages recovered by bronchoalveolar lavage were assayed for migratory response to N-formylmethlonylphenylalanlne and endotoxln-actlvated human serum. Stimulated migration of cells from healthy recipients obtained In the late postoperative course was similar to that of normal control subjects, but stimulated migration of cells from healthy recipients In the early postoperative period and those undergoing chronic rejection was greater than expected. Spontaneous migration was similar In all groups except those with P. car/nll pneumonia, In whom It was greatly Increased. We conclude that alveolar macrophage migration Is not Impaired In lung allograft recipients without apparent signs of Infection or rejection and Is In fact Increased during periods of possible macrophage activation (shortly after transplantation AM REV RESPIR DIS 1991; 143:834-838 and during chronic reJection).
patients receiveddouble-lung transplantation, one for silicosis and the other for cystic fibrosis. 1VormaIControISubjee~
Normal individuals who volunteered to undergo bronchoalveolar lavage were used as controls. These subjects werenonsmokers and had no evidence of chronic lung disease or reactive airways disease and no history of a recent respiratory tract infection in the 6 wk before lavage or exposure to fibrogenic dusts. Controls on average were 10yr younger than the patients.
Proeotol Bronchoalveolar lavage was performed at least once within the first month after transplantation, at 1- to 4-month intervals thereafter, and whenever lung infection or rejection was suspected. Normal volunteers each underwent one bronchoalveolar lavage. Informed consent for bronchoalveolar lavage was obtained from all subjects according to a protocol approved by the Institutional Review Board for Biomedical Research at the University of Pittsburgh. Severalgroups of lung recipientswereevaluated. The primary group had no evidence for rejection or infection at the time of the bronchoalveolar lavage (no infection or rejection, NIR). This group was of particular interest since we wanted to identify abnormalities of AM function in recipients who were doing
clinically well that might predispose them to pulmonary infection. Recipientswereexcluded from this group if they had clinical findings of pulmonary inflammation, such as fever and radiographic infiltrates, or if any infectious pathogens were recovered from the bronchoalveolar lavage. They were also excluded if they had ever had any evidence of rejection on lung biopsy, such as perivascular infiltrates and/or bronchiolitis obliterans, or if any additional immunosuppressive therapy had been given for suspected lung rejection within 1 wk of the bronchoalveolar lavage. In addition, none of these patients had alloreactive cells in bronchoalveolar lavage, a finding that correlates with rejection (6).
(Received in original form May 15, 1990 and in revised form September 21, 1990) 1 From the Departments of Medicine and Surgery, University of Pittsburgh School of Medicine, and the Oakland Veterans Administration Medical Center, Pittsburgh, Pennsylvania. 2 Supported by Clinical Investigator Award HL 01367and Grant No. HL 37533from the National Heart, Lung, and Blood Institute, the VeteransAdministration, and the Christmas Seal League of Western Pennsylvania. 3 Correspondence and requests for reprints should be addressed to Robert M. Hoffman, M.D., Pulmonary Division, Department of Medicine, 440 Scaife Hall, 3550 Terrace Street, Pittsburgh, PA 15261.
ALVEOLAR MACROPHAGE MIGRATION IN WNG TRANSPLANTATION
Patients who met the criteria for the NIR group were further classified as in the early posttransplant period (NIR-E) if their bronchoalveolar lavage was performed less than 6 wk after transplantation or as in the late posttransplant period (NIR-L) if their bronchoalveolar lavage was performed more than 6 wk after transplantation and there was no detectable pulmonary infection within the previous 12wk. Since6 wk is the time at which the transplanted lung appears to be nearly completely repopulated with recipient-derived AM it was chosen as the cutoff between NIR-E and NIR-L (7). Another group of patients consisted of recipients from whom cellswere obtained during a documented infection with Pneumocystis carinii (PCP) (1). The final group of patients was diagnosed as having chronic rejection (CR) of their lung allograft on the basis of bronchiolitis obliterans on transbronchial lung biopsy (8). The regimen of immunosuppression in these recipients consisted of cyclosporine A, azathioprine, and prednisone. The dose of cyclosporine A was adjusted to maintain a whole-blood level between 100 and 1,000 ng/ml by radioimmunoassay. The dose ofazathioprine was adjusted to maintain the white blood cell count above 3,500cells/rum". Prednisone was administered in a dose of 10 to 20 mg daily.
Bronchoalveolar lovage After topical lidocaine anesthesia of the oropharynx a flexible fiberoptic bronchoscope was introduced into the lungs via the nares. Lidocaine anesthesia was necessary only above the airway anastomosis for the lung transplant recipients. For the normal volunteers it was attempted to use the smallest amount of lidocaine possible to facilitate patient tolerance of the procedure. Lavage was performed through a subsegmental bronchus of the right middle lobe. Bronchoalveolar lavage was performed by injecting and aspirating four 50-ml aliquots of normal saline. Bronchoalveolar lovage Processing Bronchoalveolar lavage fluid' was filtered through two layers of surgical gauze and the volume measured. Lung lavagecellswereseparated by centrifugation (500 x g for 10min) and counted using an automatic particle counter (Coulter Electronics, Hialeah, FL) and a hemacytometer. Bronchoalveolar lavage cells were washed twice in calcium- and magnesium-free Hanks' balanced salt solution with 20 mM HEPES buffer [Grand Island Biologic Co. (GIBCO), Grand Island, NY] and resuspended in RPMI-I640 medium with L-glutamine (M.A. Bioproducts, Walkersville,MD). Cell viability was assessed by trypan blue exclusion. Cytocentrifuge slides (Cytospin" II; Shandon Southern, Sewickley,PAl were prepared for differential cell counts, which were performed using a modified Wright-Giemsa stain (Dade Diagnostics Inc., Aquada, PR).
Pneumocystis Identification Three air-dried, acetone-fixed cytocentrifuge smears of bronchoalveolar lavage cells or sections of lung tissue, when available, were stained with silver methenamine (9). These specimens wereconsidered positive for R carinii when several encysted forms were seen on at least two cytocentrifuge preparations of bronchoalveolar lavage cells or tissue sections. Migration Assay A volume of cell suspension sufficient to perform the migration assay was centrifuged and the pellet resuspended in Oey's balanced salt solution (GIBCO) containing 2010 bovine serum albumin (Sigma Chemical Company, St. Louis, MO) to give a final concentration of 2 x 1& viable alveolar macrophages per ml. This suspension was then used in the migration assay. The migration assayswereperformed in 48wellmicrochemotaxis chambers (Neuroprobe Inc, Cabin John, MD) as previouslydescribed (10). The bottom wells of the chamber were filled with 25 J.LI fluid containing the chemotactic stimulus. A polycarbonate filter with a pore size of 8 J.Lm (Nucleopore Corp., Pleasanton, CAl was placed over the bottom wells. The silicone gasket and upper piece of the chamber were applied and the entire assembly was preincubated at 37° C in humidified air for 15 min before filling the upper wells with 50 J.LI cell suspension. The chambers were then incubated at 37° C in humidified air for 2 hr. After incubation the chambers were disassembled and the filter was removed. The cells in the upper well that had not migrated through the filter were removed with a rubber policeman. The filter was then stained with a modified Wright's stain (DiffQuik il; Harleco, Philadelphia, PAl and mounted on a glass slide. Macrophages that had completely migrated through the filter were counted in 10 random oil immersion fields (OIF; x 1,(00). Migrational response was defined as the mean number of migrated macrophages per OIF for each group and expressed as cells/field. N-formylmethionylphenylalanine (FMP; Sigma) and endotoxin-activated human serum were used as the migratory stimuli in all studies. Fresh human serum was activated using the technique of Hausman and coworkers (11). A mixture of 1 part serum and 1 part Escherichia coli 0127/B8 lipopolysaccharide (Sigma), 3 mg/ml in 0.15 M phosphatebuffered saline at pH 7.2, and 3 parts gelatinveronal buffer (12) was incubated at 37° C for 60 min followed by heating at 56° C for 30 min. The stock solution of 20% endotoxinactivated human serum (vol/vol) was aliquoted and stored at - 20° C until used. For FMP a range of concentrations from 10-4 to 10-7 M was used in the migration assay. For endotoxin-activated human serum a range of dilutions from 1:2 to 1:16 was used. The experimental pitfalls of using multiwell chemotaxis chambers have recently been
835
reviewed (13). Because of the variability of the migratory response among different assays, results were normalized by expressing them in terms of a migration index: that is, the response to a given stimulus divided by the response to the negative control, multiplied by 100.
Statistical Analysis Statistical analysis consisted of a one-wayfactorial analysis of variance (ANOVA) followed by the Fischer's least significant difference method as a multiple-comparisons procedure (14). The analysis was performed with a commercially available software package (15). Group data were expressed as the mean ± So. A p value of < 0.05 wasconsideredstatistically significant.
Results There were no differences in spontaneous migration among groups except for the PCP group (6.1 ± 6.0 cells/field). This value was significantly higher than spontaneous migration seen in the normal group (0.9 ± 0.4 cells/field), the NIR-E group (0.9 ± 0.4 cells/field), the NIR-L group (1.1 ± 0.8 cells/field), and the CR group (0.8 ± 0.4 cells/field). The migratory response of AM to both stimuli was very similar throughout the course of the study, although there was considerably more intragroup variation for endotoxin-activated human serum. Therefore we have included only the data for the response to FMP in this report. Stimulated migration for the PCP group (12.4 ± 16.0 cells/field) was significantly higher than stimulated migration seen in the normal group (3.7 ± 2.3 cells/field), the NIR-L group (4.2 ± 4.7 cells/field), and the CR group (4.8 ± 3.0 cells/field). The stimulated migration for the NIR-E group (6.6 ± 4.0 cells/field) was significantly higher than for the NIR-L group and also was greater than for the normal group, although this last difference did not reach statistical significance (0.10> P > 0.05). There were no differences among the NIR-L, CR, and normal groups. For the reasons cited in Methods we chose to analyze our results by expressing them in terms of a migration index; that is, stimulated migration divided by random migration multiplied by 100. When these data were analyzed no difference was noted in the peak response to FMP expressed as the migration index between the NIR-L (385 ± 289) and the normal group (409 ± 219), but the NIR-E (785 ± 440) and CR (687 ± 434) groups showed a greater migration index than the normal group (figure 1).
HOFFMAN, DAUBER, PARADIS, GRIFFITH, AND HARDESTY
836
1800 1600 1400 1200 1000 PEAK MIGRATION INDEX 800
.. .• v
• -+-
..
600
400
~
• -+'!.
..
200
NIR-E (N-ll)
t..t:
-4L-
NIR-L (n-l7)
CR (n-13)
PCP (n=5)
NORMAL (n_13)
Fig. 1. Peak migration index of AM for FMP. See Methods for definition of migration index and patient subgroups. Data points for each patient are listed, and the mean for each group is indicated by a horizontal line. Means and SD for each group are as follows: N1R-E (785 ± 440), NIR-L (385 ± 289), CR (687 ± 434), PCP (192 ± 59), and normal (409 ± 219). Migration index for NIR-L, PCP, and normal was less than both NIR-E and CR. There was no difference between NIR-L, PCP, and normal and between NIR-E and CR.
45 40
Fig. 2. Spontaneous and peak stimulated migration of AM for PCP and CR groups. Spontaneous migration for the CR group (0.8 ± 0.4 cellslfield) was less than PCP (6.1 ± 6.0 cells/field). Stimulated migration for the CR group (4.8 ± 3.0 cells/field) was also less tr sn PCP (12.4 ± 16.0 cells/field).
35 30 MIGRATION 25 (CELLSlOtF) 20 15
....
10
.. ----*-
aneous StimJlaled
pcp
1800
NIR-E
1600 1400 1200
AlterCR
1000 PEAK MIGRATION INDEX 800 600 400 200 0 0
50
100
150
200
250
300
350
Postoperative Day
The peak migration index was actually much lower for cells from the PCP group (192 ± 59) than the NIR-E group and the CR group, but this result was expected because of the method we used to calculate the migration index [(stimulated migration/spontaneous migration) x 100]. The high level of spontaneous migration for PCP patients tended to offset the high level of stimulated migration. This is illustrated in figure 2, where the actual number of migrated alveolar macrophages is shown for spontaneous and stimulated migration for both the, PCP and CR groups. Despite the fact that the absolute value for stimulated migration is higher for PCP than CR, the migration index for the former group is much lower than that for the latter. There were 13lavages classified as CR: 5 episodes were in totally asymptomatic patients (simple bronchiolitis obliterans); 8 episodes were accompanied by a decline in the FEV 1 and increased dyspnea (complicated bronchiolitis obliterans). There was no significant difference in the peak
400
450
Fig. 3. Migratory response toward FMP for cells from a single individual who underwent serial bronchoalveolar lavage. The classification of this individual is shown at the time of each lavage. This patient illustrates the high level of stimulated migration seen for cells obtained in the early postoperative period and the decline in this function to lower levels that typically occurs later in the postoperative course.
migration index for FMP between simple (942 ± 556) and complicated (528 ± 266) bronchiolitis obliterans. Serial determinations of migration during the postoperative course in individual patients confirmed the observations made using intergroup comparisons. The peak migratory response to formyl peptide versus postoperative day in a lung transplant recipient is shown in figure 3. In the early postoperative period this patient's migration index was markedly elevated compared to normal. All subsequent values were substantially lower than this initial determination. This pattern was observed in three other patients with serial determinations (data not shown). The other notable observation in figure 3 is that despite the successful treatment of CR (elimination of active bronchiolitis on lung biopsy) with rabbit antithymocyte globulin (RATG) the patient's migration index continued to increase. This is the reason we were careful to exclude any patients who previously had bronchiolitis obliterans from the
NIR-L category, as the cells from these recipients seemed to remain activated for a prolonged time period, even after successfully treated CR. This patient then went on to experience further chronic rejection as demonstrated by transbronchial biopsy on postoperative day 525. Discussion The results of this study suggest that stimulated migration of macrophages from human lung allografts is not impaired for recipients with no obvious signs of infection or rejection (NIR). In fact, the number of macrophages capable of responding to a migratory stimulus in vitro may actually increase in the early postoperative period, when the lung allograft is being repopulated by macrophages derived from the recipient's bone marrow (7). The proportion of responsive cells may also be greater during episodes of untreated bronchiolitis obliterans, which we equate with late rejection of the allograft (16). It should be noted that the use of topical lidocaine anesthesia during bronchoscopy was more liberal in the normal volunteers than in any of the lung transplant recipients, since the recipients are essentially denervated below the airway anastomosis and do not require lidocaine once the bronchoscope is passed beyond this point. This is of some concern since lidocaine has been shown to inhibit several macrophage functions, including migration (17-19). It is conceivable that the migration measured in the normal group was decreased by lidocaine, thereby making comparisons between patients and normal subjects uninterpretable. However, the concentration of lidocaine measured in bronchoalveolar lavage is generally well below the lidocaine level required to have significant effects on macrophage function when studied in vitro (20). For this reason we believe that the higher migration index for cells from the recipients in the NIR-E and CR groups reflects an increase in the proportion of responsive cells in the lung allograft. The effect of immunosuppressive agents on the function of human alveolar macrophages has not been studed previously. The few studies performed in animals have shown inhibition of migration. Drath and Kahan (21) looked at the effect of cyclosporine A, azathioprine, and prednisolone on several functions in the rat. AM from cyclosporine A-treated animals demonstrated reduced migration
ALVEOLAR MACROPHAGE MIGRATION IN LUNG TRANSPLANTATION
toward N-formylmethionylleucylphenylalanine (FMLP). Migratory activity of normal rat AM was also inhibited by cyclosporine A in vitro. AM from rats receiving cyclosporine A and prednisolone also demonstrated decreased migratory activity toward FMLP, although to a lesser extent than observed when cyclosporine A was given alone. In contrast to the effects observed in response to FMLP, there was no inhibition of migration when zymosan-activated serum was used as the migratory stimulus. Pennington and Harris (22) showed that immunosuppression of guinea pigs with corticosteroids resulted in a 60% reduction of AM migration toward FMLP. A possible reason we did not observe these effects in our study could be the failure of the immunosuppressive agents to reach effective concentrations in the alveolar space at the dosages commonly used. Indeed, several attempts to measure the concentration of cyclosporine A by radioimmunoassay or high-performance liquid chromatography in concentrated and unconcentrated bronchoalveolar lavage fluid of lung transplant recipients failed to detect clinically significant levels (Dauber JH, Venkataramanan R, Burckart G, unpublished observations). It is planned to repeat these studies on peripheral blood monocytes obtained from patients and normal subjects in the future to more directly determine the effect of immunosuppression on migration. We have previously shown that there is a high incidence of Pneumocystis infection after heart-lung transplantation (1). All these infections occurred greater than 6 wk posttransplant and were accompanied by an increased recovery of macrophages by bronchoalveolar lavage and the apparent accumulation of activated T cells in the allograft. We show here that pulmonary macrophages obtained during Pneumocystis infection display a very high level of spontaneous migration in vitro. This may have been due to the recruitment of blood monocytes into the allograft or the activation of resident cells.The migration index was actually lower than expected in these patients, but this was due in part to the method we used to express the response to FMP [(stimulated migration/spontaneous migration) x 100]. The large value for spontaneous migration would have required a very high level of stimulated migration to cause a significant rise in the migration index. The absolute number of cells responding to the formyl pep-
tide in several of these patients was actually severalfold higher than in any of the other recipients. Since we do not have serial data on migration in any of the PCP patients, we cannot conclude with certainty that the high level of spontaneous migration was a consequence of infection, but this conclusion seems reasonable in light of the much lower level seen consistently in healthy recipients. The finding of high spontaneous migration was somewhat unanticipated since migration of macrophages recoveredduring a cellular immune response may be inhibited (23). It may reflect prior stimulation of the cells in vivo. If this were the case then future stimulation of the cells in vitro would not be expected to produce additional migration. This issue will be difficult to clarify since more intensive efforts at prophylaxis have virtually eliminated this type of infection in recipients of lung allografts at the University of Pittsburgh. The relationship between chronic rejection and migration is less clear. Patients with chronic rejection are at increased risk for acquiring bacterial infection (24), but the migratory response of their cells in vitro was greater than that of cells from healthy recipients in the late postoperative course and from normal subjects. This may be due to the accumulation of more active mononuclear cells in the alveolar space during rejection and suggests that factors other than chemotaxis predispose to infection. Even after treatment for chronic rejection the AM migratory response was likely to remain elevated despite the lack of evidence for active bronchiolitis on biopsy. The degree of elevation of the migration index was similar for recipients whose chronic rejection was symptomatic or asymptomatic. In summary we have documented that the stimulated migration of alveolar macrophages from human lung allografts fluctuates according to the clinical status of the patient, but for the most part it is unimpaired. This suggests that other factors contribute to the higher than expected frequency of bacterial pneumonia in recipients of lung-heart allografts, especially in the early postoperative period and during episodes of chronic rejection. Acknowledgment The writers thank Frank Damico for his assistance in the statistical analysis of the data.
837
The writers thank Matthew Becker and Dwayne Zern for their technical support. References 1. Gryzan S, Paradis I, Zeevi A, et al. Unexpectedly high incidence of Pneumocystis carinii infection after lung-heart transplantation: implications for lung defense and allograft survival. Am Rev Respir Dis 1988; 137:1268-74. 2. Dummer J, Montero C, Griffith B, Hardesty R, Paradis I, Ho M. Infections in heart-lung transplant recipients. Transplantation 1986; 41:725-9. 3. Dummer J, White L, Ho M, Griffith B, Hardesty R, Bahnson H. Morbidity of cytomegalovirus infection in recipients of heart or heart-lung transplants who received cyclosporine. J Infect Dis 1985; 152:1182-92. 4. Paradis I, Rabinowich H, Zeevi A, et al. Life in the allogeneic environment after lung transplantation. Lung 1990; (Suppl:1172-81). 5. Griffith B, Hardesty R, Trento A, et al. Heartlung transplantation: lessons learned and future hopes. Ann Thorac Surg 1987; 43:6-16. 6. Rabinowich H, Zeevi A, Paradis I, et al. Proliferative responses of bronchoalveolar lavage lymphocytes from heart-lung transplant patients. Transplantation 1990; 49:115-21. 7. Paradis I, Marrari M, Zeevi A, et al. HLA phenotype oflung lavage cellsfollowingheart-lung transplantation. Heart Transplant 1985; 4:422-5. 8. YousemS, Paradis I, Dauber J, Griffith B.Efficacy of transbronchiallung biopsy in the diagnosis of bronchiolitis obliterans in heart-lung transplant recipients. Transplantation 1989; 47:893-5. 9. Mahan C, Sale G. Rapid methenamine silver stain for Pneumocystis and fungi. Arch Pathol Lab Med 1978; 102:351-2. 10. Falk W, Goodwin R, Leonard E. A 48 well microchemotaxis assembly for rapid and accurate measurement of leukocyte migration. J Immunol Methods 1980; 33:239-47. 11. Hausman M, Snyderman R, Mergenhagen S. Humoral mediators of chemotaxis of mononuclear leukocytes. J Infect Dis 1972; 125:595-602. 12. Mayer M. Complement and complement fixation. In: Kabat E, Mayer M, eds. Experimental immunochemistry. Springfield, IL: Charles C. Thomas, 1961; 133-240. 13. Minkin C, Bannon D Jr, Pokress S, Melnick M. Multiwellchamber chemotaxis assays: improved experimental design and data analysis. J Immunol Methods 1985; 78:307-21. 14. Winer B. Statistical Principles in experimental design. 2nd ed. New York: McGraw-Hill, 1971. 15. Feldman D Jr, Gagnon J. StatViewS12 + Calabasas, CA: BrainPower, Inc., 1986. 16. Griffith B, Paradis I, ZeeviA, et al. Immunologically mediated disease of the airways after pulmonary transplantation. Ann Surg 1988; 208:371-8. 17. Hoidal J, White J, Repine J. Impairment of human alveolar macrophage oxygen consumption and superoxide anion production by local anesthetics used in bronchoscopy. Chest 1979; 75(Suppl.: 243-6). 18. Baser Y, deShazo R, Barkman H Jr, Nordberg J. Lidocaine effects on immunocompetent cells: implications for studies of cells obtained by bronchoalveolar lavage. Chest 1982; 82:323-8. 19. Dickstein R, Kirimidjian-Schumacher L, Stotzky G. Effect of lidocaine on production of macrophage inhibitory factor and on macrophage motility: in vitro exposure of guinea pig lymphocytes and macrophages. J Leukoc Biol 1984; 36:621-32.
HOFFMAN, DAUBER, PARADIS, GRIFFITH, AND HARDESTY
838 20. Kirkpatrick M. Lidocaine topical anesthesia for flexible bronchoscopy. Chest 1989; 96:965-6. 21. Drath D, Kahan B. Alterations in rat pulmonary macrophage function by the immunosuppressive agents cyclosporine, azathioprine, and prednisolone. Transplantation 1983; 35:588-92.
22. Pennington J, Harris E. Influence of immunosuppression on alveolar macrophage chemotaxic activities in guinea pigs. Am Rev Respir Dis 1981; 123:299-304. 23. Block L, Jaksche H, Bamberger S, Ruhenstroth-Bauer G. Human migration inhibi-
tory factor: purification and immunochemical characterization. J Exp Med 1978; 147:541-53. 24. Dauber J, Zeevi A. Pulmonary complications of lung transplantation. In: Lynch J, DeRemee R, eds. Immunologically mediated lung disease. Philadelphia: J. B. Lippincott (In Press).
ROBERT M. HOFFMAN, JAMES H. DAUBER, IRVIN L. PARADIS, BARTLEY P. GRIFFITH, and ROBERT L. HARDESTY
Introduction
Pulmonary infection has been a major source of morbidity and mortality in all immunosuppressed organ transplant recipients, but in recipients of a lung allograft it is particularly troublesome (1-3). The factors responsible for the higher than expectedfrequency and mortality are unclear at present, but defective pulmonary host defense mechanisms of the lung allograft probably playa key role. The alveolar macrophage (AM) is the resident phagocytic cell in the lung, and it is responsible for maintaining sterility in the distal airspaces. However, its function may be abnormal in the transplanted lung not only because of the effects of immunosuppressive drugs and infection, but also because it exists in a potentially hostile allogeneic environment (4). In this study AM were recovered from bronchoalveolar lavage performed on lung transplant recipients and assayedfor migratory activity. We elected to study this particular function because it is thought to be important in the early response to the deposition of bacteria in the distal airspaces of the lung. Wecompared AM migration between normal individuals and lung allograft recipients who weresubclassified according to their clinical status. In addition, the migratory activity of AM was tracked in a sequential fashion so that variations in migration within an individual patient could be correlated with that patient's clinical status. Methods Lung Transplant Recipients Lung transplant recipients were followed prospectively between July 1984 and May 1989. Of the 28 patients in the study 26 received lung and heart allografts for primary or secondary pulmonary hypertension refractory to medical therapy. Patients with secondary hypertension had intracardiac shunts or, in one case, pulmonary eosinophilic granuloma (5). Because the emphasis of this report is on the lung allograft, these patients are referred to as lung transplant recipients. 1\vo 834
SUMMARY Pulmonary Infection Is a major source of morbidity and mortality In recipients of lung allografts. The alveolar macrophage plays an Important role In pulmonary host defense, and to fulfill this role It must have the ability to orient and migrate In the direction of a stimulus. Thus migratory activity was measured In cells recovered from lung transplant recipients by bronchoalveolar lavage. The primary patient group consisted of recipients who had no evidence of Infection or rejection at the time of bronchoalveolar lavage. These patients were further subdlvldad Into an early postoperative group (less than 6 wk posttransplant) and a late postoperative group (greater than 6 wk posttransplant). Other categories Included patients with chronic rejection and a small group of patients with Pneumocystls car/nll pneumonia. Alveolar macrophages recovered by bronchoalveolar lavage were assayed for migratory response to N-formylmethlonylphenylalanlne and endotoxln-actlvated human serum. Stimulated migration of cells from healthy recipients obtained In the late postoperative course was similar to that of normal control subjects, but stimulated migration of cells from healthy recipients In the early postoperative period and those undergoing chronic rejection was greater than expected. Spontaneous migration was similar In all groups except those with P. car/nll pneumonia, In whom It was greatly Increased. We conclude that alveolar macrophage migration Is not Impaired In lung allograft recipients without apparent signs of Infection or rejection and Is In fact Increased during periods of possible macrophage activation (shortly after transplantation AM REV RESPIR DIS 1991; 143:834-838 and during chronic reJection).
patients receiveddouble-lung transplantation, one for silicosis and the other for cystic fibrosis. 1VormaIControISubjee~
Normal individuals who volunteered to undergo bronchoalveolar lavage were used as controls. These subjects werenonsmokers and had no evidence of chronic lung disease or reactive airways disease and no history of a recent respiratory tract infection in the 6 wk before lavage or exposure to fibrogenic dusts. Controls on average were 10yr younger than the patients.
Proeotol Bronchoalveolar lavage was performed at least once within the first month after transplantation, at 1- to 4-month intervals thereafter, and whenever lung infection or rejection was suspected. Normal volunteers each underwent one bronchoalveolar lavage. Informed consent for bronchoalveolar lavage was obtained from all subjects according to a protocol approved by the Institutional Review Board for Biomedical Research at the University of Pittsburgh. Severalgroups of lung recipientswereevaluated. The primary group had no evidence for rejection or infection at the time of the bronchoalveolar lavage (no infection or rejection, NIR). This group was of particular interest since we wanted to identify abnormalities of AM function in recipients who were doing
clinically well that might predispose them to pulmonary infection. Recipientswereexcluded from this group if they had clinical findings of pulmonary inflammation, such as fever and radiographic infiltrates, or if any infectious pathogens were recovered from the bronchoalveolar lavage. They were also excluded if they had ever had any evidence of rejection on lung biopsy, such as perivascular infiltrates and/or bronchiolitis obliterans, or if any additional immunosuppressive therapy had been given for suspected lung rejection within 1 wk of the bronchoalveolar lavage. In addition, none of these patients had alloreactive cells in bronchoalveolar lavage, a finding that correlates with rejection (6).
(Received in original form May 15, 1990 and in revised form September 21, 1990) 1 From the Departments of Medicine and Surgery, University of Pittsburgh School of Medicine, and the Oakland Veterans Administration Medical Center, Pittsburgh, Pennsylvania. 2 Supported by Clinical Investigator Award HL 01367and Grant No. HL 37533from the National Heart, Lung, and Blood Institute, the VeteransAdministration, and the Christmas Seal League of Western Pennsylvania. 3 Correspondence and requests for reprints should be addressed to Robert M. Hoffman, M.D., Pulmonary Division, Department of Medicine, 440 Scaife Hall, 3550 Terrace Street, Pittsburgh, PA 15261.
ALVEOLAR MACROPHAGE MIGRATION IN WNG TRANSPLANTATION
Patients who met the criteria for the NIR group were further classified as in the early posttransplant period (NIR-E) if their bronchoalveolar lavage was performed less than 6 wk after transplantation or as in the late posttransplant period (NIR-L) if their bronchoalveolar lavage was performed more than 6 wk after transplantation and there was no detectable pulmonary infection within the previous 12wk. Since6 wk is the time at which the transplanted lung appears to be nearly completely repopulated with recipient-derived AM it was chosen as the cutoff between NIR-E and NIR-L (7). Another group of patients consisted of recipients from whom cellswere obtained during a documented infection with Pneumocystis carinii (PCP) (1). The final group of patients was diagnosed as having chronic rejection (CR) of their lung allograft on the basis of bronchiolitis obliterans on transbronchial lung biopsy (8). The regimen of immunosuppression in these recipients consisted of cyclosporine A, azathioprine, and prednisone. The dose of cyclosporine A was adjusted to maintain a whole-blood level between 100 and 1,000 ng/ml by radioimmunoassay. The dose ofazathioprine was adjusted to maintain the white blood cell count above 3,500cells/rum". Prednisone was administered in a dose of 10 to 20 mg daily.
Bronchoalveolar lovage After topical lidocaine anesthesia of the oropharynx a flexible fiberoptic bronchoscope was introduced into the lungs via the nares. Lidocaine anesthesia was necessary only above the airway anastomosis for the lung transplant recipients. For the normal volunteers it was attempted to use the smallest amount of lidocaine possible to facilitate patient tolerance of the procedure. Lavage was performed through a subsegmental bronchus of the right middle lobe. Bronchoalveolar lavage was performed by injecting and aspirating four 50-ml aliquots of normal saline. Bronchoalveolar lovage Processing Bronchoalveolar lavage fluid' was filtered through two layers of surgical gauze and the volume measured. Lung lavagecellswereseparated by centrifugation (500 x g for 10min) and counted using an automatic particle counter (Coulter Electronics, Hialeah, FL) and a hemacytometer. Bronchoalveolar lavage cells were washed twice in calcium- and magnesium-free Hanks' balanced salt solution with 20 mM HEPES buffer [Grand Island Biologic Co. (GIBCO), Grand Island, NY] and resuspended in RPMI-I640 medium with L-glutamine (M.A. Bioproducts, Walkersville,MD). Cell viability was assessed by trypan blue exclusion. Cytocentrifuge slides (Cytospin" II; Shandon Southern, Sewickley,PAl were prepared for differential cell counts, which were performed using a modified Wright-Giemsa stain (Dade Diagnostics Inc., Aquada, PR).
Pneumocystis Identification Three air-dried, acetone-fixed cytocentrifuge smears of bronchoalveolar lavage cells or sections of lung tissue, when available, were stained with silver methenamine (9). These specimens wereconsidered positive for R carinii when several encysted forms were seen on at least two cytocentrifuge preparations of bronchoalveolar lavage cells or tissue sections. Migration Assay A volume of cell suspension sufficient to perform the migration assay was centrifuged and the pellet resuspended in Oey's balanced salt solution (GIBCO) containing 2010 bovine serum albumin (Sigma Chemical Company, St. Louis, MO) to give a final concentration of 2 x 1& viable alveolar macrophages per ml. This suspension was then used in the migration assay. The migration assayswereperformed in 48wellmicrochemotaxis chambers (Neuroprobe Inc, Cabin John, MD) as previouslydescribed (10). The bottom wells of the chamber were filled with 25 J.LI fluid containing the chemotactic stimulus. A polycarbonate filter with a pore size of 8 J.Lm (Nucleopore Corp., Pleasanton, CAl was placed over the bottom wells. The silicone gasket and upper piece of the chamber were applied and the entire assembly was preincubated at 37° C in humidified air for 15 min before filling the upper wells with 50 J.LI cell suspension. The chambers were then incubated at 37° C in humidified air for 2 hr. After incubation the chambers were disassembled and the filter was removed. The cells in the upper well that had not migrated through the filter were removed with a rubber policeman. The filter was then stained with a modified Wright's stain (DiffQuik il; Harleco, Philadelphia, PAl and mounted on a glass slide. Macrophages that had completely migrated through the filter were counted in 10 random oil immersion fields (OIF; x 1,(00). Migrational response was defined as the mean number of migrated macrophages per OIF for each group and expressed as cells/field. N-formylmethionylphenylalanine (FMP; Sigma) and endotoxin-activated human serum were used as the migratory stimuli in all studies. Fresh human serum was activated using the technique of Hausman and coworkers (11). A mixture of 1 part serum and 1 part Escherichia coli 0127/B8 lipopolysaccharide (Sigma), 3 mg/ml in 0.15 M phosphatebuffered saline at pH 7.2, and 3 parts gelatinveronal buffer (12) was incubated at 37° C for 60 min followed by heating at 56° C for 30 min. The stock solution of 20% endotoxinactivated human serum (vol/vol) was aliquoted and stored at - 20° C until used. For FMP a range of concentrations from 10-4 to 10-7 M was used in the migration assay. For endotoxin-activated human serum a range of dilutions from 1:2 to 1:16 was used. The experimental pitfalls of using multiwell chemotaxis chambers have recently been
835
reviewed (13). Because of the variability of the migratory response among different assays, results were normalized by expressing them in terms of a migration index: that is, the response to a given stimulus divided by the response to the negative control, multiplied by 100.
Statistical Analysis Statistical analysis consisted of a one-wayfactorial analysis of variance (ANOVA) followed by the Fischer's least significant difference method as a multiple-comparisons procedure (14). The analysis was performed with a commercially available software package (15). Group data were expressed as the mean ± So. A p value of < 0.05 wasconsideredstatistically significant.
Results There were no differences in spontaneous migration among groups except for the PCP group (6.1 ± 6.0 cells/field). This value was significantly higher than spontaneous migration seen in the normal group (0.9 ± 0.4 cells/field), the NIR-E group (0.9 ± 0.4 cells/field), the NIR-L group (1.1 ± 0.8 cells/field), and the CR group (0.8 ± 0.4 cells/field). The migratory response of AM to both stimuli was very similar throughout the course of the study, although there was considerably more intragroup variation for endotoxin-activated human serum. Therefore we have included only the data for the response to FMP in this report. Stimulated migration for the PCP group (12.4 ± 16.0 cells/field) was significantly higher than stimulated migration seen in the normal group (3.7 ± 2.3 cells/field), the NIR-L group (4.2 ± 4.7 cells/field), and the CR group (4.8 ± 3.0 cells/field). The stimulated migration for the NIR-E group (6.6 ± 4.0 cells/field) was significantly higher than for the NIR-L group and also was greater than for the normal group, although this last difference did not reach statistical significance (0.10> P > 0.05). There were no differences among the NIR-L, CR, and normal groups. For the reasons cited in Methods we chose to analyze our results by expressing them in terms of a migration index; that is, stimulated migration divided by random migration multiplied by 100. When these data were analyzed no difference was noted in the peak response to FMP expressed as the migration index between the NIR-L (385 ± 289) and the normal group (409 ± 219), but the NIR-E (785 ± 440) and CR (687 ± 434) groups showed a greater migration index than the normal group (figure 1).
HOFFMAN, DAUBER, PARADIS, GRIFFITH, AND HARDESTY
836
1800 1600 1400 1200 1000 PEAK MIGRATION INDEX 800
.. .• v
• -+-
..
600
400
~
• -+'!.
..
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NIR-E (N-ll)
t..t:
-4L-
NIR-L (n-l7)
CR (n-13)
PCP (n=5)
NORMAL (n_13)
Fig. 1. Peak migration index of AM for FMP. See Methods for definition of migration index and patient subgroups. Data points for each patient are listed, and the mean for each group is indicated by a horizontal line. Means and SD for each group are as follows: N1R-E (785 ± 440), NIR-L (385 ± 289), CR (687 ± 434), PCP (192 ± 59), and normal (409 ± 219). Migration index for NIR-L, PCP, and normal was less than both NIR-E and CR. There was no difference between NIR-L, PCP, and normal and between NIR-E and CR.
45 40
Fig. 2. Spontaneous and peak stimulated migration of AM for PCP and CR groups. Spontaneous migration for the CR group (0.8 ± 0.4 cellslfield) was less than PCP (6.1 ± 6.0 cells/field). Stimulated migration for the CR group (4.8 ± 3.0 cells/field) was also less tr sn PCP (12.4 ± 16.0 cells/field).
35 30 MIGRATION 25 (CELLSlOtF) 20 15
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10
.. ----*-
aneous StimJlaled
pcp
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AlterCR
1000 PEAK MIGRATION INDEX 800 600 400 200 0 0
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Postoperative Day
The peak migration index was actually much lower for cells from the PCP group (192 ± 59) than the NIR-E group and the CR group, but this result was expected because of the method we used to calculate the migration index [(stimulated migration/spontaneous migration) x 100]. The high level of spontaneous migration for PCP patients tended to offset the high level of stimulated migration. This is illustrated in figure 2, where the actual number of migrated alveolar macrophages is shown for spontaneous and stimulated migration for both the, PCP and CR groups. Despite the fact that the absolute value for stimulated migration is higher for PCP than CR, the migration index for the former group is much lower than that for the latter. There were 13lavages classified as CR: 5 episodes were in totally asymptomatic patients (simple bronchiolitis obliterans); 8 episodes were accompanied by a decline in the FEV 1 and increased dyspnea (complicated bronchiolitis obliterans). There was no significant difference in the peak
400
450
Fig. 3. Migratory response toward FMP for cells from a single individual who underwent serial bronchoalveolar lavage. The classification of this individual is shown at the time of each lavage. This patient illustrates the high level of stimulated migration seen for cells obtained in the early postoperative period and the decline in this function to lower levels that typically occurs later in the postoperative course.
migration index for FMP between simple (942 ± 556) and complicated (528 ± 266) bronchiolitis obliterans. Serial determinations of migration during the postoperative course in individual patients confirmed the observations made using intergroup comparisons. The peak migratory response to formyl peptide versus postoperative day in a lung transplant recipient is shown in figure 3. In the early postoperative period this patient's migration index was markedly elevated compared to normal. All subsequent values were substantially lower than this initial determination. This pattern was observed in three other patients with serial determinations (data not shown). The other notable observation in figure 3 is that despite the successful treatment of CR (elimination of active bronchiolitis on lung biopsy) with rabbit antithymocyte globulin (RATG) the patient's migration index continued to increase. This is the reason we were careful to exclude any patients who previously had bronchiolitis obliterans from the
NIR-L category, as the cells from these recipients seemed to remain activated for a prolonged time period, even after successfully treated CR. This patient then went on to experience further chronic rejection as demonstrated by transbronchial biopsy on postoperative day 525. Discussion The results of this study suggest that stimulated migration of macrophages from human lung allografts is not impaired for recipients with no obvious signs of infection or rejection (NIR). In fact, the number of macrophages capable of responding to a migratory stimulus in vitro may actually increase in the early postoperative period, when the lung allograft is being repopulated by macrophages derived from the recipient's bone marrow (7). The proportion of responsive cells may also be greater during episodes of untreated bronchiolitis obliterans, which we equate with late rejection of the allograft (16). It should be noted that the use of topical lidocaine anesthesia during bronchoscopy was more liberal in the normal volunteers than in any of the lung transplant recipients, since the recipients are essentially denervated below the airway anastomosis and do not require lidocaine once the bronchoscope is passed beyond this point. This is of some concern since lidocaine has been shown to inhibit several macrophage functions, including migration (17-19). It is conceivable that the migration measured in the normal group was decreased by lidocaine, thereby making comparisons between patients and normal subjects uninterpretable. However, the concentration of lidocaine measured in bronchoalveolar lavage is generally well below the lidocaine level required to have significant effects on macrophage function when studied in vitro (20). For this reason we believe that the higher migration index for cells from the recipients in the NIR-E and CR groups reflects an increase in the proportion of responsive cells in the lung allograft. The effect of immunosuppressive agents on the function of human alveolar macrophages has not been studed previously. The few studies performed in animals have shown inhibition of migration. Drath and Kahan (21) looked at the effect of cyclosporine A, azathioprine, and prednisolone on several functions in the rat. AM from cyclosporine A-treated animals demonstrated reduced migration
ALVEOLAR MACROPHAGE MIGRATION IN LUNG TRANSPLANTATION
toward N-formylmethionylleucylphenylalanine (FMLP). Migratory activity of normal rat AM was also inhibited by cyclosporine A in vitro. AM from rats receiving cyclosporine A and prednisolone also demonstrated decreased migratory activity toward FMLP, although to a lesser extent than observed when cyclosporine A was given alone. In contrast to the effects observed in response to FMLP, there was no inhibition of migration when zymosan-activated serum was used as the migratory stimulus. Pennington and Harris (22) showed that immunosuppression of guinea pigs with corticosteroids resulted in a 60% reduction of AM migration toward FMLP. A possible reason we did not observe these effects in our study could be the failure of the immunosuppressive agents to reach effective concentrations in the alveolar space at the dosages commonly used. Indeed, several attempts to measure the concentration of cyclosporine A by radioimmunoassay or high-performance liquid chromatography in concentrated and unconcentrated bronchoalveolar lavage fluid of lung transplant recipients failed to detect clinically significant levels (Dauber JH, Venkataramanan R, Burckart G, unpublished observations). It is planned to repeat these studies on peripheral blood monocytes obtained from patients and normal subjects in the future to more directly determine the effect of immunosuppression on migration. We have previously shown that there is a high incidence of Pneumocystis infection after heart-lung transplantation (1). All these infections occurred greater than 6 wk posttransplant and were accompanied by an increased recovery of macrophages by bronchoalveolar lavage and the apparent accumulation of activated T cells in the allograft. We show here that pulmonary macrophages obtained during Pneumocystis infection display a very high level of spontaneous migration in vitro. This may have been due to the recruitment of blood monocytes into the allograft or the activation of resident cells.The migration index was actually lower than expected in these patients, but this was due in part to the method we used to express the response to FMP [(stimulated migration/spontaneous migration) x 100]. The large value for spontaneous migration would have required a very high level of stimulated migration to cause a significant rise in the migration index. The absolute number of cells responding to the formyl pep-
tide in several of these patients was actually severalfold higher than in any of the other recipients. Since we do not have serial data on migration in any of the PCP patients, we cannot conclude with certainty that the high level of spontaneous migration was a consequence of infection, but this conclusion seems reasonable in light of the much lower level seen consistently in healthy recipients. The finding of high spontaneous migration was somewhat unanticipated since migration of macrophages recoveredduring a cellular immune response may be inhibited (23). It may reflect prior stimulation of the cells in vivo. If this were the case then future stimulation of the cells in vitro would not be expected to produce additional migration. This issue will be difficult to clarify since more intensive efforts at prophylaxis have virtually eliminated this type of infection in recipients of lung allografts at the University of Pittsburgh. The relationship between chronic rejection and migration is less clear. Patients with chronic rejection are at increased risk for acquiring bacterial infection (24), but the migratory response of their cells in vitro was greater than that of cells from healthy recipients in the late postoperative course and from normal subjects. This may be due to the accumulation of more active mononuclear cells in the alveolar space during rejection and suggests that factors other than chemotaxis predispose to infection. Even after treatment for chronic rejection the AM migratory response was likely to remain elevated despite the lack of evidence for active bronchiolitis on biopsy. The degree of elevation of the migration index was similar for recipients whose chronic rejection was symptomatic or asymptomatic. In summary we have documented that the stimulated migration of alveolar macrophages from human lung allografts fluctuates according to the clinical status of the patient, but for the most part it is unimpaired. This suggests that other factors contribute to the higher than expected frequency of bacterial pneumonia in recipients of lung-heart allografts, especially in the early postoperative period and during episodes of chronic rejection. Acknowledgment The writers thank Frank Damico for his assistance in the statistical analysis of the data.
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The writers thank Matthew Becker and Dwayne Zern for their technical support. References 1. Gryzan S, Paradis I, Zeevi A, et al. Unexpectedly high incidence of Pneumocystis carinii infection after lung-heart transplantation: implications for lung defense and allograft survival. Am Rev Respir Dis 1988; 137:1268-74. 2. Dummer J, Montero C, Griffith B, Hardesty R, Paradis I, Ho M. Infections in heart-lung transplant recipients. Transplantation 1986; 41:725-9. 3. Dummer J, White L, Ho M, Griffith B, Hardesty R, Bahnson H. Morbidity of cytomegalovirus infection in recipients of heart or heart-lung transplants who received cyclosporine. J Infect Dis 1985; 152:1182-92. 4. Paradis I, Rabinowich H, Zeevi A, et al. Life in the allogeneic environment after lung transplantation. Lung 1990; (Suppl:1172-81). 5. Griffith B, Hardesty R, Trento A, et al. Heartlung transplantation: lessons learned and future hopes. Ann Thorac Surg 1987; 43:6-16. 6. Rabinowich H, Zeevi A, Paradis I, et al. Proliferative responses of bronchoalveolar lavage lymphocytes from heart-lung transplant patients. Transplantation 1990; 49:115-21. 7. Paradis I, Marrari M, Zeevi A, et al. HLA phenotype oflung lavage cellsfollowingheart-lung transplantation. Heart Transplant 1985; 4:422-5. 8. YousemS, Paradis I, Dauber J, Griffith B.Efficacy of transbronchiallung biopsy in the diagnosis of bronchiolitis obliterans in heart-lung transplant recipients. Transplantation 1989; 47:893-5. 9. Mahan C, Sale G. Rapid methenamine silver stain for Pneumocystis and fungi. Arch Pathol Lab Med 1978; 102:351-2. 10. Falk W, Goodwin R, Leonard E. A 48 well microchemotaxis assembly for rapid and accurate measurement of leukocyte migration. J Immunol Methods 1980; 33:239-47. 11. Hausman M, Snyderman R, Mergenhagen S. Humoral mediators of chemotaxis of mononuclear leukocytes. J Infect Dis 1972; 125:595-602. 12. Mayer M. Complement and complement fixation. In: Kabat E, Mayer M, eds. Experimental immunochemistry. Springfield, IL: Charles C. Thomas, 1961; 133-240. 13. Minkin C, Bannon D Jr, Pokress S, Melnick M. Multiwellchamber chemotaxis assays: improved experimental design and data analysis. J Immunol Methods 1985; 78:307-21. 14. Winer B. Statistical Principles in experimental design. 2nd ed. New York: McGraw-Hill, 1971. 15. Feldman D Jr, Gagnon J. StatViewS12 + Calabasas, CA: BrainPower, Inc., 1986. 16. Griffith B, Paradis I, ZeeviA, et al. Immunologically mediated disease of the airways after pulmonary transplantation. Ann Surg 1988; 208:371-8. 17. Hoidal J, White J, Repine J. Impairment of human alveolar macrophage oxygen consumption and superoxide anion production by local anesthetics used in bronchoscopy. Chest 1979; 75(Suppl.: 243-6). 18. Baser Y, deShazo R, Barkman H Jr, Nordberg J. Lidocaine effects on immunocompetent cells: implications for studies of cells obtained by bronchoalveolar lavage. Chest 1982; 82:323-8. 19. Dickstein R, Kirimidjian-Schumacher L, Stotzky G. Effect of lidocaine on production of macrophage inhibitory factor and on macrophage motility: in vitro exposure of guinea pig lymphocytes and macrophages. J Leukoc Biol 1984; 36:621-32.
HOFFMAN, DAUBER, PARADIS, GRIFFITH, AND HARDESTY
838 20. Kirkpatrick M. Lidocaine topical anesthesia for flexible bronchoscopy. Chest 1989; 96:965-6. 21. Drath D, Kahan B. Alterations in rat pulmonary macrophage function by the immunosuppressive agents cyclosporine, azathioprine, and prednisolone. Transplantation 1983; 35:588-92.
22. Pennington J, Harris E. Influence of immunosuppression on alveolar macrophage chemotaxic activities in guinea pigs. Am Rev Respir Dis 1981; 123:299-304. 23. Block L, Jaksche H, Bamberger S, Ruhenstroth-Bauer G. Human migration inhibi-
tory factor: purification and immunochemical characterization. J Exp Med 1978; 147:541-53. 24. Dauber J, Zeevi A. Pulmonary complications of lung transplantation. In: Lynch J, DeRemee R, eds. Immunologically mediated lung disease. Philadelphia: J. B. Lippincott (In Press).
