Adhesion Molecules (E-Selectin and ICAM-1) in Pulmonary Allograft Rejection
1996; Elsevier BV; Volume: 110; Issue: 5 Linguagem: Inglês
10.1378/chest.110.5.1143
ISSN1931-3543
AutoresRevati Shreeniwas, Larry L. Schulman, Mangala Narasimhan, Carlton C. McGregor, Charles C. Marboe,
Tópico(s)Mast cells and histamine
ResumoVascular endothelial cells act as antigen-presenting cells in the lung allograft and stimulate alloreactive host lymphocytes. Activated lymphocytes and cytokines can induce expression of leukocyte-endothelial adhesion molecules that facilitate invasion of the allograft by circulating leukocytes. To define the role of endothelial HLA class II antigen and adhesion molecule expression in lung allograft rejection, we prospectively analyzed endothelial expression of HLA class II, E-selectin, and intercellular adhesion molecule-1 (ICAM-1) antigens in 52 transbronchial biopsy specimens from 24 lung allograft recipients as compared to normal control subjects. Thirty-one of 52 specimens showed histologic rejection and 8 of 24 patients developed histologic obliterative bronchiolitis (OB) by the end of the study period. Increased expression of HLA class II antigen was seen in 32 of 52 (62%) lung allograft specimens, but increased expression did not correlate with acute rejection or OB. In contrast, E-selectin expression was seen in 30 of 52 (58%) biopsy specimens and was associated with acute rejection (p<0.005) and with the development of OB (p<0.05). Increased expression of ICAM-1 was seen in only 18 of 52 (35%) biopsy specimens and did not correlate with acute rejection or OB. These data suggest that E-selectin expression may be a tissue marker of acute and chronic lung rejection possibly by promoting leukocyte adhesion to the allograft endothelium. The high levels of endothelial HLA class II expression may reflect long-term antigenic stimulation of the allograft even in the absence of rejection. Vascular endothelial cells act as antigen-presenting cells in the lung allograft and stimulate alloreactive host lymphocytes. Activated lymphocytes and cytokines can induce expression of leukocyte-endothelial adhesion molecules that facilitate invasion of the allograft by circulating leukocytes. To define the role of endothelial HLA class II antigen and adhesion molecule expression in lung allograft rejection, we prospectively analyzed endothelial expression of HLA class II, E-selectin, and intercellular adhesion molecule-1 (ICAM-1) antigens in 52 transbronchial biopsy specimens from 24 lung allograft recipients as compared to normal control subjects. Thirty-one of 52 specimens showed histologic rejection and 8 of 24 patients developed histologic obliterative bronchiolitis (OB) by the end of the study period. Increased expression of HLA class II antigen was seen in 32 of 52 (62%) lung allograft specimens, but increased expression did not correlate with acute rejection or OB. In contrast, E-selectin expression was seen in 30 of 52 (58%) biopsy specimens and was associated with acute rejection (p<0.005) and with the development of OB (p<0.05). Increased expression of ICAM-1 was seen in only 18 of 52 (35%) biopsy specimens and did not correlate with acute rejection or OB. These data suggest that E-selectin expression may be a tissue marker of acute and chronic lung rejection possibly by promoting leukocyte adhesion to the allograft endothelium. The high levels of endothelial HLA class II expression may reflect long-term antigenic stimulation of the allograft even in the absence of rejection. cytomegalovirus intercellular adhesion molecule-1 obliterative bronchiolitis phosphate-buffered saline solution transbronchial biopsy In lung transplantation as a therapeutic option for severe pulmonary disease, there is a high incidence of acute and chronic rejection that may be more frequent as compared with other organ allografts.1Griffith BP Hardesty RL Trento A et al.Heart-lung transplantation: lessons learned and future hopes.Ann Thorac Surg. 1987; 43: 6-16Abstract Full Text PDF PubMed Scopus (161) Google Scholar, 2Grossman RF Cooper JD. Lung transplantation.Immunol Allergy Clin North Am. 1989; 9: 153-163Google Scholar, 3Theodore J Lewiston N. Lung transplantation comes of age.N Engl J Med. 1990; 322: 727-733Crossref PubMed Scopus (63) Google Scholar Although acute allograft rejection episodes are common, the major cause of long-term morbidity is the development of obliterative bronchiolitis (OB), which is generally considered to represent chronic allograft rejection.4Cooper JD Patterson GA Grossman R et al.Double lung transplant for chronic obstructive pulmonary disease.Am Rev Respir Dis. 1989; 139: 303-307Crossref PubMed Scopus (155) Google Scholar, 5LoCicero J Robinson PG Fisher M. Chronic rejection in single lung transplantation as manifested by obliterative bronchiolitis.J Thorac Cardiovasc Surg. 1990; 99: 1059-1062PubMed Google Scholar, 6Sharpies LD Tamm M McNeil K et al.Development of bronchiolitis obliterans syndrome in recipients of heart-lung transplantation—early risk factors.Transplantation. 1996; 61: 560-566Crossref PubMed Scopus (149) Google Scholar Recurrent episodes of acute rejection and infection have been implicated in the pathogenesis of OB.7Scott JP Higginbottam TW Clelland CA et al.Natural history of chronic rejection in heart-lung transplant recipients.J Heart Transplant. 1990; 9: 510-515PubMed Google Scholar, 8Yousem SA Burke CM Billingham ME. Pathologic pulmonary alterations in long-term human heart-lung transplantation.Hum Pathol. 1985; 16: 911-923Abstract Full Text PDF PubMed Scopus (172) Google ScholarTable 2Contingency Analyses*Contingency tables showing the number of biopsy specimens from lung allografts in each category. Tissue was processed and stained for HLA class II antigen, E-selectin, and ICAM-1. Positive staining and histologic rejection were determined as described in the text.* Contingency tables showing the number of biopsy specimens from lung allografts in each category. Tissue was processed and stained for HLA class II antigen, E-selectin, and ICAM-1. Positive staining and histologic rejection were determined as described in the text. Open table in a new tab Acute lung rejection is characterized by perivascular and mucosal inflammatory cell infiltrates.8Yousem SA Burke CM Billingham ME. Pathologic pulmonary alterations in long-term human heart-lung transplantation.Hum Pathol. 1985; 16: 911-923Abstract Full Text PDF PubMed Scopus (172) Google Scholar, 9Tazelaar HD. Perivascular inflammation in pulmonary infections: implications for the diagnosis of lung rejection.J Heart Lung Transplant. 1991; 10: 437-441PubMed Google Scholar, 10Higginbottam T Stewart S Penketh A et al.Transbronchial lung biopsy for the diagnosis of rejection in heart-lung transplant recipients.Transplantation. 1988; 46: 532-539Crossref PubMed Scopus (186) Google Scholar HLA class II antigens are known to be involved in the rejection response11Van Seventer GA Shimizu Y Shaw S. Roles of multiple accessory molecules in T-cell activation.Curr Opin Immunol. 1991; 3: 294303Crossref Scopus (156) Google Scholar, 12Milton AD Fabre JW. Massive induction of donor type class I and class II histocompatibility complex antigens in rejecting cardiac allografts in the rat.J Exp Med. 1985; 161: 98-112Crossref PubMed Scopus (176) Google Scholar, 13Fries JWU Williams AJ Atkins RC et al.Expression of VCAM-1 and E-selectin in an in vivo model of endothelial activation.Am J Pathol. 1993; 143: 725-737PubMed Google Scholar in part by vascular endothelial cells acting as antigen-presenting cells. Increased expression of class II antigens on the endothelial surface can stimulate alloreactive T lymphocytes, inducing a local inflammatory response. Activated lymphocytes and cytokines in conjunction with activated vascular endothelium in the allografted lung initiate the process of acute rejection.14Yousem SA Curley JM Dauber J et al.HLA class II antigen expression in human heart-lung allografts.Transplantation. 1990; 49: 991-995Crossref PubMed Scopus (50) Google Scholar Though capillary endothelium from normal lung tissue can express low levels of HLA class II antigens,15DeMeester Sr., Rolfe MW Kunkel SL et al.The bimodal expression of tumor necrosis factor-α in association with rat lung reimplantation and allograft rejection.J Immunol. 1993; 150: 24942505Google Scholar there are little data regarding upregulation of class II antigen expression and pulmonary allograft rejection. In the acute rejection process, activated alveolar macrophages and cytokine-primed T lymphocytes in conjunction with cytokines can stimulate endothelial cells, inducing expression of several adhesion molecules, such as E-selectin, and intercellular adhesion molecule-1 (ICAM-1).16Briscoe DM Yeung AC Schoen FJ et al.Predictive value of inducible endothelial cell adhesion molecule expression for acute rejection of human cardiac allografts.Transplantation. 1995; 59: 204-211Crossref PubMed Scopus (66) Google Scholar These adhesion molecules mediate leukocyte-endothelial interactions17Carlos T Kovach N Schwartz B et al.Human monocytes bind to two cytokine induced adhesive ligands on cultured human endothelial cells: ELAM-1 and VCAM-1.Blood. 1991; 77: 2260-2271Google Scholar, 18Picker LJ Kishimoto TK Smith TW et al.ELAM-1 is an adhesion molecule for skin homing T cells.Nature. 1991; 349: 796-799Crossref PubMed Scopus (742) Google Scholar, 19Shimizu Y Shaw S Graber N et al.Activation-independent binding of human memory T cells to adhesion molecule ELAM-1.Nature. 1991; 349: 799-802Crossref PubMed Scopus (320) Google Scholar, 20Osborn L. Leukocyte adhesion to endothelium in inflammation.Cell. 1990; 62: 3-6Abstract Full Text PDF PubMed Scopus (780) Google Scholar, 21McEver RP. Leukocyte-endothelial cell interactions.Curr Opin Cell Biol. 1992; 4: 840-849Crossref PubMed Scopus (159) Google Scholar and may facilitate invasion of the allograft by circulating leukocytes, further amplifying the local inflammatory response. E-selectin is a representative of a novel class of adhesion molecules called selectins.22Lasky LA. Selectins; interpreters of cell specific carbohydrate information during inflammation.Science. 1992; 258: 964-969Crossref PubMed Scopus (1153) Google Scholar, 23Pober JS. Cytokine-mediated activation of vascular endothelium: physiology and pathology.Am J Pathol. 1988; 133: 426-433PubMed Google Scholar, 24Bevilacqua MP Stengelin S Gimbrone MA et al.Endothelial leukocyte adhesion molecule-1:an inducible receptor for neutrophils related to complement regulatory proteins and lectins.Science. 1989; 243: 1160-1165Crossref PubMed Scopus (1682) Google Scholar Both in vivo as well as in cell culture, this molecule is not expressed on resting endothelium.25Seekamp A Warren JS Remick DG et al.Requirements for tumor necrosis factor-α and interleukin-1 in limb ischemia/reperfusion injury and associated lung injury.Am J Pathol. 1993; 143: 453-463PubMed Google Scholar, 26Wegner CD Gundel RH Rothlein R et al.Expression and probable roles of cell adhesion molecules in lung inflammation.Chest. 1992; 101: 34S-39SAbstract Full Text Full Text PDF PubMed Google Scholar E-selectin is induced on endothelium by cytokines such as interleukin-1 and tumor necrosis factor-a and also by direct contact with primed CD4+ T cells. In endothelial cell monolayers in culture, it is rapidly induced, within 4 h following cytokine stimulation and is transiently expressed, returning to baseline after 8 h.19Shimizu Y Shaw S Graber N et al.Activation-independent binding of human memory T cells to adhesion molecule ELAM-1.Nature. 1991; 349: 799-802Crossref PubMed Scopus (320) Google Scholar, 20Osborn L. Leukocyte adhesion to endothelium in inflammation.Cell. 1990; 62: 3-6Abstract Full Text PDF PubMed Scopus (780) Google Scholar, 24Bevilacqua MP Stengelin S Gimbrone MA et al.Endothelial leukocyte adhesion molecule-1:an inducible receptor for neutrophils related to complement regulatory proteins and lectins.Science. 1989; 243: 1160-1165Crossref PubMed Scopus (1682) Google Scholar E-selectin binds to sialated Lewis X glycoproteins on the surface of leukocytes27Brandley DK Sweidler SJ Robbins PW. Carbohydrate ligands of the LEC cell adhesion molecules.Cell. 1990; 63: 861-863Abstract Full Text PDF PubMed Scopus (294) Google Scholar and mediates the adhesion of polymorphonuclear leukocytes, monocytes, and some memory T cells to the surface of activated endothelium.20Osborn L. Leukocyte adhesion to endothelium in inflammation.Cell. 1990; 62: 3-6Abstract Full Text PDF PubMed Scopus (780) Google Scholar, 21McEver RP. Leukocyte-endothelial cell interactions.Curr Opin Cell Biol. 1992; 4: 840-849Crossref PubMed Scopus (159) Google Scholar, 22Lasky LA. Selectins; interpreters of cell specific carbohydrate information during inflammation.Science. 1992; 258: 964-969Crossref PubMed Scopus (1153) Google Scholar, 23Pober JS. Cytokine-mediated activation of vascular endothelium: physiology and pathology.Am J Pathol. 1988; 133: 426-433PubMed Google Scholar, 24Bevilacqua MP Stengelin S Gimbrone MA et al.Endothelial leukocyte adhesion molecule-1:an inducible receptor for neutrophils related to complement regulatory proteins and lectins.Science. 1989; 243: 1160-1165Crossref PubMed Scopus (1682) Google Scholar ICAM-1 (CD54) is a member of the immunoglobulin gene superfamily.28Dustin ML Rothlein R Bhan AK et al.Induction by II-1 and Interferon gamma: tissue distribution, biochemistry, and function of a natural adherence molecule (ICAM-1).J Immunol. 1986; 137: 245-254PubMed Google Scholar Though constitutively expressed by endothelium, epithelium, and macrophages, its level of expression is increased by cytokine stimulation.20Osborn L. Leukocyte adhesion to endothelium in inflammation.Cell. 1990; 62: 3-6Abstract Full Text PDF PubMed Scopus (780) Google Scholar, 29Tosi MF Stark JM Smith CW et al.Induction of ICAM-1 expression on human airway epithelial cells by inflammatory cytokines: effects on neutrophil-epithelial cell adhesion.Am J Respir Cell Mol Biol. 1992; 7: 214-220Crossref PubMed Scopus (249) Google Scholar In endothelial cell monolayers in culture, increased expression of ICAM-1 is seen beginning at 6 h after cytokine stimulation and levels stay elevated for periods up to 24 h.21McEver RP. Leukocyte-endothelial cell interactions.Curr Opin Cell Biol. 1992; 4: 840-849Crossref PubMed Scopus (159) Google Scholar, 26Wegner CD Gundel RH Rothlein R et al.Expression and probable roles of cell adhesion molecules in lung inflammation.Chest. 1992; 101: 34S-39SAbstract Full Text Full Text PDF PubMed Google Scholar, 28Dustin ML Rothlein R Bhan AK et al.Induction by II-1 and Interferon gamma: tissue distribution, biochemistry, and function of a natural adherence molecule (ICAM-1).J Immunol. 1986; 137: 245-254PubMed Google Scholar, 29Tosi MF Stark JM Smith CW et al.Induction of ICAM-1 expression on human airway epithelial cells by inflammatory cytokines: effects on neutrophil-epithelial cell adhesion.Am J Respir Cell Mol Biol. 1992; 7: 214-220Crossref PubMed Scopus (249) Google Scholar The ligands for ICAM-1 on the leukocyte are the integrins LFA-1 and Mac-1. Interaction between LFA-1 on T lymphocytes with ICAM-1 on antigen presenting cells has been shown to be costimulatory in activation of the T-cell receptor complex and could potentially amplify the population of activated T lymphocytes in the allograft.11Van Seventer GA Shimizu Y Shaw S. Roles of multiple accessory molecules in T-cell activation.Curr Opin Immunol. 1991; 3: 294303Crossref Scopus (156) Google Scholar In this preliminary study, we prospectively analyzed endothelial expression of HLA class II antigen expression in transbronchial biopsy specimens from lung transplant recipients to determine the level of antigenic stimulation in the allograft. We also analyzed expression of E-selectin and ICAM-1 in the same tissue in an attempt to identify tissue markers of rejection and possibly predict the subsequent development of obliterative bronchiolitis (OB). Twenty-four lung transplant recipients underwent 52 transbronchial biopsy (TBB) procedures from 1991 to 1993. Twenty-three of the 52 bronchoscopies were performed for clinical indications (suspicion of infection or rejection), and 29 were performed for routine surveillance which was done every 3 months in the absence of symptoms. Mean timing of biopsy with respect to date of transplant surgery was 401 (SD, 358; range, 10 to 1,815) days. Mean number of biopsy episodes subjected to immunohistochemistry was 3.2 (SD, 1.9; range, 1 to 6) biopsy episodes per patient. All patients received a standard immunosupressive regimen of cyclosporine, azathioprine, and prednisone. Five patients who underwent TBBs for evaluation of solitary nodules or lung masses provided samples for control lung tissue. Biopsies were performed in a radiographically normal area adjacent to the lesion. All control patients were former smokers. The bronchoscopic procedure was performed under local anesthesia by the transnasal approach. TBBs were performed using alligator forceps (Olympus FB-15C; Olympus Corp; Lake Success, NY) under fluoroscopic guidance from multiple subsegments of a lower lobe in the study group, and as described in the control group. At least six and usually eight specimens were obtained. Biopsy specimens were fixed in formaldehyde solution and processed for routine histopathologic study. One or two biopsy specimens were embedded in OCT compound (10.24% w/v polyvinylchloride, 4.26% w/v polyethelyne glycol, 85.5% nonreactive ingredients; Miles Inc, Elkhart, Ind) and frozen for immunohistochemical evaluation. Rejection was graded according to the standard criteria set forth by the Lung Rejection Study Group.30Yousem SA Berry GJ Brunt EM et al.A working formulation for the standardization of nomenclature in the diagnosis of heart lung rejection: Lung Rejection Study Group.J Heart Transplant. 1990; 2: 593-601Google Scholar Antibody to ICAM-1 is a mouse monoclonal antibody RR.1 and was used at a concentration of 5 μg/mL. Antibody to E-selectin is a mouse monoclonal antibody 3B7 and was used at a concentration of 5 μg/mL. Antibody to HLA class II antigen is a mouse monoclonal against the β chain of the DR complex and was used at a concentration of 5 μg/mL. One or two biopsy specimens of adequate size were subjected to immunohistochemical staining at each time point. Ten-micronthick frozen sections were washed with 0.3% hydrogen peroxide in methanol for 15 min to block endogenous tissue peroxidase, and blocked with 1% horse serum in phosphate-buffered saline solution (PBS) for 30 min. The sections were then incubated with the primary antibody overnight at 4°C, washed with PBS, and incubated with biotinylated horse antimouse IgG (PK 6102 Vectastain Elite Kit; Vector Labs; Burlinghame, Calif). Color development was with 3,3' diaminobenzidine 0.6 mg/mL in 0.1 mol/L Tris-saline solution buffer with 0.005% hydrogen peroxide for 10 min and counter staining was with hematoxylin. To determine background staining for every sample, tissue sections were incubated overnight with mouse IgG of the same subclass as the monoclonal antibodies in question diluted 1:10 in PBS instead of the primary antibody. In each instance, at least three to five sections from a biopsy piece were examined. One or two biopsy specimens were examined at each time point. To minimize day-to-day variability in immunohistochemical staining, positive and negative controls were run each time a batch of TBB specimens was stained. Stained sections were examined in a blinded manner by one of us (C.C.M.). To quantify antigen expression, sections were scored on the basis of intensity of focal staining and the percentage of included vessels in a given field. At least five high-power fields were counted, and the mean score was taken. Based on this approach, the biopsy specimens were graded as follows: 0, absence of staining; basal, faint staining on 0 to 25% of vessels per high-power field; and increased, intense staining on more than 25% of all vessels per high-power field. Positive staining was correlated to the presence of rejection by contingency analysis and significance tested by Fisher's Exact Test. Multivariate analysis was performed using a stepwise logistic regression model.31Bishop YMM. Full contingency tables, logits, and split contingency tables.J Biometrics. 1969; 25: 383-389Crossref Google Scholar In no case did the logistic regression analysis give a different result from the univariate model. Fifty-two biopsy specimens from 24 lung transplant recipients were studied. Thirty-one of 52 (60%) allograft specimens showed evidence of histologic rejection. Eight of the 24 patients developed OB (proved by histologic study) by the end of the 15-month study period. In addition, three patients developed a clinical OB syndrome by the end of the study period. Three of the 11 patients had OB at the time of biopsy. In lung tissue samples from the control group (n=5), there was no E-selectin seen (absent staining). In one of five cases there was low-level (basal) staining for HLA class II antigen on the capillary endothelium, increased in one of five cases, and absent in three of five. In four of five cases there was low-level (basal) of staining of ICAM-1 and absent in one of five (Table 1). This pattern of expression of HLA class II antigen, E-selectin, and ICAM-1 on uninflamed endothelium agrees with previous reports in the literature.21McEver RP. Leukocyte-endothelial cell interactions.Curr Opin Cell Biol. 1992; 4: 840-849Crossref PubMed Scopus (159) Google Scholar, 22Lasky LA. Selectins; interpreters of cell specific carbohydrate information during inflammation.Science. 1992; 258: 964-969Crossref PubMed Scopus (1153) Google Scholar, 25Seekamp A Warren JS Remick DG et al.Requirements for tumor necrosis factor-α and interleukin-1 in limb ischemia/reperfusion injury and associated lung injury.Am J Pathol. 1993; 143: 453-463PubMed Google Scholar, 30Yousem SA Berry GJ Brunt EM et al.A working formulation for the standardization of nomenclature in the diagnosis of heart lung rejection: Lung Rejection Study Group.J Heart Transplant. 1990; 2: 593-601Google Scholar We therefore considered increased expression of HLA class II antigen, E-selectin, and ICAM-1 in the study group to include those allograft samples that displayed greater than basal level of staining for ICAM-1 and HLA class II antigen and any level of staining for E-selectin.Table 1Expression of HLA Class II Antigens and Adhesion Molecules (E-Selectin and ICAM-1) in TBB Specimens Antigen GradeControl (%) (n=5)Allograft (%) (n=52)HLA class IIAbsent3/5 (60) 4/52 (8)Basal1/5 (20)16/52 (31)Increased1/5 (20)32/52 (62)E-selectinAbsent5/5 (100)22/52 (42)Increased0/5 (0)30/52 (58)ICAM-1Absent1/5 (20)11/52 (21)Basal4/5 (80)23/52 (44)Increased0/5 (0)18/52 (35) Open table in a new tab Increased expression of HLA class II antigen was seen in 32 of 52 (62%) biopsy specimens. There was no staining for HLA class II antigen in 4 of 52 (8%) allografts and basal staining in 16 of 52 (31%) (Table 1). Increased HLA class II antigen expression did not correlate with acute rejection in that increased staining was seen on vascular endothelium in 17 of 31 (55%) allografts with rejection and in 15 of 21 (71%) allografts without rejection. In addition, there was no correlation between increased HLA class II antigen expression and the development of OB. An example of increased staining for HLA class II antigen expression in an allograft with acute rejection is seen in Figure 1, top. Basal levels of staining are seen in an allograft without rejection (Fig 1, bottom). E-selectin expression was observed in 30 of 52 (58%) lung allograft specimens (Table 1). E-selectin expression was seen in 23 of 31 rejections (74% sensitivity) and in 7 of 21 nonrejecting tissue samples (67% specificity; Table 2, panel A). The odds ratio for E-selectin expression in predicting acute rejection was 5.75 (p<0.005). There was no significant difference noted in the pattern of staining on bronchial or pulmonary vessels. There was, however, an increased number of E-selectin positive vessels near lymphoid aggregates. An example of increased expression of E-selectin on capillaiy endothelium in an allograft with acute rejection is shown in Figure 2, top. Absent staining for E-selectin in a nonrejecting allograft is shown in Figure 2, bottom. In three patients, initial biopsy specimens showed E-selectin expression, but tissue taken at later time points did not show staining for E-selectin, at which time in all three instances there was no evidence of acute rejection on histologic study. In one patient, increased expression of E-selectin was seen in the face of acute rejection, but on an earlier and on a later biopsy specimen where there was no evidence of rejection, no E-selectin staining was seen. One patient with persistent rejection had increased expression of E-selectin seen at six time points; she had histologic rejection in four of six time points. There was an association between E-selectin expression on vascular endothelium and the development of histologic OB (Table 2, panel B), with a sensitivity of 100% and a specificity of 50% (p<0.05). When clinical OB and histologic OB were considered together as a single entity (Table 2, panel C), this relationship with E-selectin was maintained (sensitivity, 91%; specificity, 61%; p<0.1). E-selectin expression was seen concomitantly with histologic OB in three of eight patients and preceded manifestations in the three patients who developed clinical OB. Increased expression of ICAM-1 was noted in 18 of 52 (35%) biopsy specimens. Eleven of 52 (21%) allografts had no staining for ICAM-1 and basal expression was seen on 23 of 52 (44%) allografts (Table 2). When present, staining was seen in capillary endothelium, alveolar epithelium, alveolar macrophages, and lymphocytes. Tissue samples that showed staining in nonendothelial cells showed concomitant staining in endothelial cells. There was no difference in the staining of bronchial and pulmonary vessels. There was increased ICAM-1 staining adjacent to lymphocyte aggregates. Unlike E-selectin, increased endothelial expression of ICAM-1 did not correlate with acute rejection or with the development of OB. Increased endothelial staining for ICAM-1 was seen in 11 of 31 (36%) allografts with rejection, and in 7 of 21 (33%) allografts without rejection. An example of increased staining for ICAM-1 in an allograft with acute rejection is shown in Figure 3, top. An example of basal staining for ICAM-1 in allograft lung without rejection is shown in Figure 3, bottom. There was overlap in the pattern of expression of HLA class II antigen and ICAM-1 (Table 2, panel D), and biopsy specimens that had increased expression of ICAM-1 also had increased expression of HLA class II antigen (p<0.005; odds ratio, 9). There was no correlation between the expression of E-selectin and HLA class II antigen. Fifteen of the 52 bronchoscopies had cytomegalovirus (CMV) cultured from the BAL fluid (CMV infection), but none of the biopsy sections had inclusion bodies characteristic of CMV pneumonitis. There was no correlation between CMV infection and histologic rejection, nor was there any correlation between CMV infection and increased E-selectin or ICAM-1 expression or the development of OB. There was a single instance of mild Pneumocystis carinii infection. Foamy alveolar exudates and organisms were seen in the section stained for adhesion molecule expression. In this instance, E-selectin expression was absent. In lung allografts, the vascular endothelium is the main interface between immunologically active recipient leukocytes and the donor lung. Donor HLA class II antigens expressed on the allograft endothelium are recognized by the T-cell receptor complex on circulating lymphocytes prior to initiation of specific immune reactions. Inducible adhesion molecules such as E-selectin and ICAM-1 may play an important role in the initiation of the acute rejection process and ultimately in the development of OB. In our study, we analyzed transbronchial biopsy specimens from lung allografts and found that there was upregulation of class II antigen expression on vascular endothelium that was present even in the absence of rejection. However, we found that there was an association between E-selectin expression and acute rejection as well as the subsequent development of OB. We did not find an association between increased ICAM-1 expression and rejection, although there was overlap between the pattern of expression of ICAM-1 and HLA class II antigen expression. Ligand binding to major histocompatibility complex class II molecules has been shown to activate leukocyte function-associated antigen-1 function, thereby providing a reciprocal mechanism for the stimulation of T cells by antigen presenting cells,32Mourad W Geha RS Chatila T. Engagement of major histocompatibility complex class II molecules induces sustained, lymphocyte function-associated molecule-1 dependent adhesion.J Exp Med. 1990; 172: 1513-1516Crossref PubMed Scopus (152) Google Scholar, 33Springer TA. Adhesion receptors of the immune system.Nature. 1990; 346: 425-434Crossref PubMed Scopus (5857) Google Scholar possibly via ICAM-1. The increased HLA class II antigen expression in the allograft specimens examined suggests ongoing antigenic stimulation34Marboe CC Buffaloe A Fenoglio JJ. Immunologic aspects of rejection.Prog Cardiovasc Dis. 1990; 6: 419-432Abstract Full Text PDF Scopus (16) Google Scholar in the allograft even in the absence of rejection. However, although enhanced ICAM-1 expression was associated with increased expression of HLA class II antigen, this pattern of ICAM-1 expression on the allograft endothelium did not correlate with either acute rejection or OB. One reason for this may be that ICAM-1 is constitutively expressed on the surface of vascular endothelium, and immunohistochemical techniques may be too insensitive to detect subtle increases in expression of ICAM-1. Another consideration may be that ICAM-1 is shed from the surface of activated endothelium. Other adhesion molecules such as vascular cell adhesion molecule-1 (CD106)17Carlos T Kovach N Schwartz B et al.Human monocytes bind to two cytokine induced adhesive ligands on cultured human endothelial cells: ELAM-1 and VCAM-1.Blood. 1991; 77: 2260-2271Google Scholar, 18Picker LJ Kishimoto TK Smith TW et al.ELAM-1 is an adhesion molecule for skin homing T cells.Nature. 1991; 349: 796-799Crossref PubMed Scopus (742) Google Scholar, 19Shimizu Y Shaw S Graber N et al.Activation-independent binding of human memory T cells to adhesion molecule ELAM-1.Nature. 1991; 349: 799-802Crossref PubMed Scopus (320) Google Scholar, 20Osborn L. Leukocyte adhesion to endothelium in inflammation.Cell. 1990; 62: 3-6Abstract Full Text PDF PubMed Scopus (780) Google Scholar, 21McEver RP. Leukocyte-endothelial cell interactions.Curr Opin Cell Biol. 1992; 4: 840-849Crossref PubMed Scopus (159) Google Scholar, 22Lasky LA. Selectins; interpreters of cell specific carbohydrate information during inflammation.Science. 1992; 258: 964-969Crossref PubMed Scopus (1153) Google Scholar have been shown to be associated with allograft rejection in other organ systems and are also costimulatory in T-cell activation, and will be studied in pulmonary allografts to see if vascular cell adhesion molecule-1 expression rather than ICAM-1 expression is related to acute lung rejection. E-selectin plays a central role in the cascade of leukocyte adhesion to endothelium.20Osborn L. Leukocyte adhesion to endothelium in inflammation.Cell. 1990; 62: 3-6Abstract Full Text PDF PubMed Scopus (780) Google Scholar, 21McEver RP. Leukocyte-endothelial cell interactions.Curr Opin Cell Biol. 1992; 4: 840-849Crossref PubMed Scopus (159) Google Scholar, 22Lasky LA. Selectins; interpreters of cell specific carbohydrate information during inflammation.Science. 1992; 258: 964-969Crossref PubMed Scopus (1153) Google Scholar, 23Pober JS. Cytokine-mediated activation of vascular endothelium: physiology and pathology.Am J Pathol. 1988; 133: 426-433PubMed Google Scholar, 24Bevilacqua MP Stengelin S Gimbrone MA et al.Endothelial leukocyte adhesion molecule-1:an inducible receptor for neutrophils related to complement regulatory proteins and lectins.Science. 1989; 243: 1160-1165Crossref PubMed Scopus (1682) Google Scholar, 25Seekamp A Warren JS Remick DG et al.Requirements for tumor necrosis factor-α and interleukin-1 in limb ischemia/reperfusion injury and associated lung injury.Am J Pathol. 1993; 143: 453-463PubMed Google Scholar, 26Wegner CD Gundel RH Rothlein R et al.Expression and probable roles of cell adhesion molecules in lung inflammation.Chest. 1992; 101: 34S-39SAbstract Full Text Full Text PDF PubMed Google Scholar It is hypothesized that the initial "rolling" leukocyte adhesion with a selectin is a prerequisite for stable adhesion to vascular endothelium under conditions of flow.26Wegner CD Gundel RH Rothlein R et al.Expression and probable roles of cell adhesion molecules in lung inflammation.Chest. 1992; 101: 34S-39SAbstract Full Text Full Text PDF PubMed Google Scholar, 27Brandley DK Sweidler SJ Robbins PW. Carbohydrate ligands of the LEC cell adhesion molecules.Cell. 1990; 63: 861-863Abstract Full Text PDF PubMed Scopus (294) Google Scholar Our data show that E-selectin is expressed during acute rejection episodes. Such expression may promote leukocyte adherence to endothelium and facilitate invasion of the allograft by circulating leukocytes. Alternatively, E-selectin expression may be a result of endothelial stimulation by cytokines such as interleukin-3 released by activated T lymphocytes during acute rejection.35Brizzi MF Garbarino G Rossi PR et al.Interleukin-3 stimulates proliferation and triggers ELAM-1 gene activation of human endothelial cells.J Clin Invest. 1993; 91: 2887-2892Crossref PubMed Scopus (76) Google Scholar In either case, expression of E-selectin in a TBB specimen is associated with acute rejection. OB represents a significant source of morbidity and mortality in lung transplant recipients.7Scott JP Higginbottam TW Clelland CA et al.Natural history of chronic rejection in heart-lung transplant recipients.J Heart Transplant. 1990; 9: 510-515PubMed Google Scholar, 8Yousem SA Burke CM Billingham ME. Pathologic pulmonary alterations in long-term human heart-lung transplantation.Hum Pathol. 1985; 16: 911-923Abstract Full Text PDF PubMed Scopus (172) Google Scholar Endothelial inflammation in the allograft could lead to disruption of the vascular barrier leading to infiltration of tissue by activated T lymphocytes, which can secrete mitogenic substances, including tumor necrosis factor and platelet-derived growth factor. These mediators have been implicated in the fibroproliferative and inflammatory response seen in OB,36Hertz MI Henke CA Nahkleh RF et al.Obliterative bronchiolitis after lung transplantation: a fibroproliferative disorder associated with platelet-derived growth factor.Proc Natl Acad Sci USA. 1992; 89: 10385-10389Crossref PubMed Scopus (152) Google Scholar but a tissue marker predicting the subsequent development of OB has not been defined (to our knowledge). We correlated E-selectin expression in tissue with histologic OB to examine tissue expression in this setting. Our data show that E-selectin staining in the vascular endothelium is closely associated with the subsequent development of OB, and positive staining for E-selectin may have a potential predictive value in this context. In addition, E-selectin expression in tissue also correlated with clinical OB in three instances. However, our data show that while the sensitivity of tissue staining for E-selectin was high, the specificity and negative predictive value of E-selectin was low. This would preclude its use as a diagnostic assay for pulmonary allograft rejection or OB. Several studies have shown that endothelial adhesion molecules are involved in kidney,37Cosimi AB Conti D Delmonico FL et al.In vivo effects of monoclonal antibody to ICAM-1 (CD54) in non human primates with renal allografts.J Immunol. 1990; 144: 4604-4612PubMed Google Scholar liver,38Adams DH Hubscher SG Shaw J et al.Intercellular adhesion molecule-1 in liver allograft during rejection.Lancet. 1989; 2: 1122-1125Abstract PubMed Scopus (204) Google Scholar and cardiac39Allen MD, McDonald TO Carlos T et al.Endothelial adhesion molecules in heart transplantation.J Heart Lung Transplant. 1992; 11: S8-13PubMed Google Scholar, 40Briscoe DM Schoen FJ Rice GE et al.Induced expression of endothelial leukocyte adhesion molecules in human cardiac allografts.Transplantation. 1991; 51: 537-547Crossref PubMed Scopus (140) Google Scholar rejection. In concordance with these studies, our data suggest that E-selectin staining may have a predictive value in identifying an episode of acute rejection as well as separating a subset of patients at risk for developing OB. However, there are other environmental influences in the lung that could influence E-selectin expression, such as regional hypoxia41Shreeniwas R Koga S Karakurum M et al.Hypoxia mediated induction of endothelial cell interleukin-lα: an autocrine mechanism promoting expression of leukocyte adhesion molecules on the vessel surface.J Clin Invest. 1992; 90: 23333-23339Crossref Scopus (310) Google Scholar and bacterial products such as lipopolysaccharide.13Fries JWU Williams AJ Atkins RC et al.Expression of VCAM-1 and E-selectin in an in vivo model of endothelial activation.Am J Pathol. 1993; 143: 725-737PubMed Google Scholar In addition, E-selectin may be locally expressed in response to infection.42Sanchez LA Rogers K Marboe CC. Endothelial leukocyte adhesion molecule-1 expression in human lung allografts.J Heart Lung Transplant. 1992; 111: 94Google Scholar In a small survey of nontransplant lung biopsy specimens, there was focal endothelial staining for E-selectin in the region of P carinii infection and CMV pneumonitis. In our study, there was only a single instance of mild P carinii and no cases of CMV pneumonitis. The histologic picture of rejection and the detection of antigens in the allografted lung tissue are influenced by sampling error in TBB specimens. It should also be noted that our control group consisted of persons who had been exposed to tobacco smoke, which may have an effect on cytokine release in the lung and HLA class II antigen expression. These clinical and technical problems, which are inherent in dealing with small samples of TBB tissue, preclude broad generalizations of our data and dictate the need for further investigation in a larger study. Despite these limitations, our data suggest that E-selectin expression on endothelium in the lung allograft may be associated with the pathophysiologic condition of acute and chronic rejection. The authors thank Dr. D. Zanger for help with data collection, Dr R. Sciacca for help with statistical analyses, and Drs. Peter Caldwell, Yale Enson, and Jane Morse for their helpful comments. Dr. Robert Rothlein, Boerhinger Ingelheim, Ridgefield, Conn, provided the antibody to ICAM-1. Dr. Barry Wolitzky, Hoffman LaRoche Inc, Nutley, NJ, provided the antibody to E-selectin. Dr. Soldano Ferrone, New York Medical College, Valhalla, NY, provided the antibody to HLA class antigen.
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