Immunohistologic characterization of synovial membrane lymphocytes in rheumatoid arthritis.код для вставкиСкачать
32 IMMUNOHISTOLOGIC CHARACTERIZATION OF SYNOVIAL MEMBRANE LYMPHOCYTES IN RHEUMATOID ARTHRITIS CAROL I,. YOUNG, THOMAS C. ADAMSON, Ill, JOHN H. VAUGHAN, and ROBERT I . FOX Synovial membrane biopsy specimens from 15 rheumatoid arthritis patients were examined using routine histologic stains and monoclonal antibodies directed against cell surface antigens. Three patterns of lymphoid cell infiltrates were recognized: 1) diffuse infiltration of T cells that surrounded clusters of germinal center B cells (3 patients); 2) diffuse T cell infiltration, lacking germinal centers (8 patients); and 3) proliferation of subsynovial fibroblasts, with relatively few lymphoid cells (4 patients). The synovial, subsynovial, and perivascular tissues in each of the patterns exhibited a high frequency of HLA-DR antigen, HLA-DS antigen, transferrin receptor, and/or epidermal growth factor receptor. In contrast, normal or osteoarthritic synovial tissues did not display a marked increase of these antigens or receptors. Cells bearing natural killer antigen were infrequent in each of these patterns. Active synovitis, synovial effusions, anemia, and elevated sedimentation rate were present in rheumatoid arthritis patients with each of the three histologic patterns. Immunohistologic characterization of synovial mem- . -. From the Department of Basic and Clinical Research. Scripps Clinic and Research Foundation. La Jolla. California. This work was supported by NIH grants AM 21 175 and CA 28746. Publication number 2941UCR from the Research Institute of Scripps Clinic. Carol I*. Young, MD: Thomas C. Adamson. 111. MD: current address: Rees-Stealy Medical Group. 2001 Fourth Avenue, San Diego. CA 9’2101: John 11. Vaughan. MD; Robert Fox, MD, PhD. Address reprint requests to Kobert I. Fox, MD. Scripps Clinic and Research Foundation, 10666 North Torrey Pines Road. BCR4. La Jolla, CA 92037. Submitted for publication June 10. 1983: accepted in revised form August 15, 1983. Arthritis and Rheumatism, Vol. 27, No. 1 (January 1984) brane infiltrates by these monoclonal antibodies provides additional information about pathogenesis of rheumatoid arthritis and may help in predicting responses to different therapeutic modalities. When many rheumatoid arthritis (RA) patients first see a rheumatologist, they have a history of longstanding activc disease that has previously been treated with multiple medications. Their responses to subsequent medications are quite variable, and laboratory tests to predict prognosis or response to therapy would aid in disease management for individual patients. As an initial step in the achievement of these aims and to better understand pathogenesis of R A , we have used monoclonal antibodies and immunohistologic techniques to characterize the mononuclear cells infiltrating the synovial membranes. Recent studies have used monoclonal antibodies directed against T cell subsets and HLA-DR antigens in frozen tissue sections of synovial membrane (1-6). We wished to expand these studies by examining tissue sections for additional cell surface markers on T cells and B cells that may be present in a patient’s synovial tissue but not necessarily present in the blood or synovial fluid. We have also used newly developed antibodies that detect antigens on synovial lining cells and vascular endothelial cells (anti-transferrin receptor and anti-epidermal growth factor receptor antibodies). These antibodies provide information about the site of inflammation not available from the study of blood or synovial fluid lymphocytes. 33 SYNOVIAL MEMBRANE LYMPHOCYTES IN RA Table 1. Clinical and laboratory data for I5 rheumatoid arthritis (RA) patients* Synovial membrane pattern Number of patients/ number of KFi patients 2 3 312 81.5 414 Total or mean 2 SD I5/l I 1 KF titer 1:1.920 c YO5 Westergrcn ESK. mmi hour 68 = 43 4.088 5 I .83n 65 .! 1 :3,055 c 2.888 66 I x3.776 I :2.720 A 66 + + Hemoglobin. gmidl Age at onset RA 20 48 11.6 :!-2.0 11.8’ 1.9 12.3 I 0.7 64 49 33 11.9 48 L 1.5 37 5 I0 Duration of RA (years)+ Slow-acting agent history$. X I 8 2 6 12 + 14 13 2 I I 14 5 5 + 14 I?I 9 -+ 9 4 * RF = rheumatoid factor. calculated for patients who were seropositive: ESR = erythrocyte sedimentation rate t Years from onset RA to surgery for synovial membrane biopsy. t Number of patients treated with gold, D-penicillamine. azathioprine, and/or cyclophosphamide. PATIENTS AND METHODS Patient population. Synovial tissue was obtained from 15 patients with definite or classic rheumatoid arthritis who underwent synovectomy or total joint replacement at Scripps Clinic from July 1980 through June 1987. Their clinical characteristics and laboratory findings are summarized in Table I . Briefly, RA patients with each of the three patterns of lymphoid cell infiltrates had active synovitis manifested by joint swelling. pain, stiffness. and recurrent synovial effusions containing elevated white cell counts and protein content (7). Slow-acting agents (gold. penicillamine. and/or azathioprine) and intraarticular steroids had been used in patients with each pattern. Synovial membrane samples used as controls were obtained from 8 patients with osteoarthritis at the time of joint replacement and from 2 patients with previously normal joints, undergoing surgery for knee trauma. Preparation of synovial membrane samples. Synovial tissue, obtained at the time of surgery. was embedded in optimal temperature cutting compound (Miles Laboratories, Naperville. IL). oriented to assure cross sections containing the synoviocyte layer, frozen using a mixture of isopentanel dry ice, and stored at -70°C (8). Multiple samples from ditrerent regions of each synovium were chosen for study, since it is known that the histologic appearance can vary from site to site within the joint (9.10). ‘Ihe particular pattern (i.e.. pattern I , 2 , or 3) was assigned on the basis of evaluation of multiple frozen tissue and paraffin-embedded specimens taken from different regions of the joint. A separate portion of tissue was digested with collagensse (Sigma, St. Louis, MO) for 16 hours at 37°C to obtain cell suspensions. This was followed by centrifugation over Ficoll-Hypaque to obtain mononuclear cells ( I I ) . Monoclonal antibodies. Monoclonal antibodies used in this study included anti-’l’ cell antibodies Leu-Za. Leu-3a, Leu-4 (Becton-Dickinson, Mountain View, CA) (17). In normal peripheral blood, suppressor/cytotoxic cells are included in the Leu-2a subset and helpcr/inducer cells in the Leu-3a subset (12). However. it is not possible to simply equate lymphocyte phenotype and function ( 1 3-17). Monoclonal antibody B532 (a gift from Dr. Dennis Frisman at University of California, San Diego) reacts with B cells found in the germinal centers of normal lymph nodes and tonsils but not with peripheral blood B cells (8,181 or synovial fluid B cells (Young CIS: unpublished observations). Two-color immunofluorescence staining studies have shown that B532’ cells bear surface immunoglobulin, and in vitro functional studies have demonstrated that B532 ’ cells synthesize immunoglobulin after mitogen stimulation in the presence of ’I cells. Antibody F45 has been characterized in our laboratory as reacting with the same subset of natural killer cells as antibody HNK-I (19). based on two-color immunofluorescence staining methods and in vitio functional analysis (Fox RI. Fraenkel F: unpublished observations). Antibody L22 reacts with the transferrin receptor. since it immunoprecipitates the same 90,000 dalton molecule as antibody B3/25 described by Trowbridge and Omary (20) (Fox RI, Dillman R: unpublished observations). Monoclonal antibodies SC2 and ,I+eu-lO (Becton-Dickinson)‘detect cell surface HLA-DK and HLA-DS antigens, respectively (7.21). Antibodies SC2. L22. and F45 are produced in our laboratory. Monoclonal antibody 455 to epidermal growth factor receptor, received from Dr. John Mendelson ( 2 2 ) .was selected for its ability to inhibit the binding of purified epidermal growth factor to the A43 1 adenocarcinoma cell line. Myeloma proteins MOPC-21. GPC7, and HO-22, with no known anti-human activity. were used as negative controls. Enumeration of lymphocyte subsets by immunohistologic and cytofluorometric analysis. Serial sections of frozen synovial membrane were stained with mouse monoclonal antibody Leu-2a. Leu-3a. Leu-4, or control myeloma protein, followed by biotin conjugated goat F(ab’)? anti-mouse IgG (’fago, Burlingame, CA), avidin conjugated horse radish peroxidase (Vector, Burlingame. CA). and substrate diaminobenzidine (DAB) (Sigma) (8). Because of their more intense staining characteristics. antibody SCZ, Leu-10, L22. 455, and F45 could be used with peroxidase conjugated F(ab’)? anti-mouse Ig (‘I‘ago) and DAB. Coded slides were read by at least two different observers. Cell suspensions derived from synovial membranes were incubated with monoclonal antibody and fluorescein-conjugated F(ab’)2anti-mouse Ig, followed by cytofluorometric analysis (7) using a FACS IV (Becton-Dickinson). 34 YOUNG ET AL RESULTS Immunohistology of RA synovial membrane. Three histologic patterns of RA synovial membrane, based on the distribution of lymphoid cell infiltrates, were observed. Pattern 1, characterized by presence of germinal ccnters, was seen in biopsy specimens from 3 of 15 patients. Figure 1A illustrates a hematoxylin and eosin stained RA synovial membrane section that contains a germinal center. Lymphoid cells in these germinal centers were stained with monoclonal antibody B532 (Figure I B), using the immunopcroxidasc technique. Leu-4 ' T cells (Figure 1 D) were located predominant- Figure 2. Synovial membrane with pattern 2 infiltrate was stained with: A. Hematoxylin-eosin; B, Antibody B532; C. Control protein MOPC-21; D. Antibody I,cu-4. ly around the germinal centers and were more numer- ous than the B cells. No significant staining was noted Figure 1. Frozen tissue sections of rheumatoid arthritis synovial membrane with pattern 1 infiltrate. A, A germinal center (arrow) detected by hematoxylin-eosin stain. B, C, D, Irnmunoperoxidase technique has been used to stain serial tissue sections: B,Antibody B532' cells within the germinal center; D,T cells (antibody Leu-4.') surrounding the germinal center. C, A control myeloma antibody (MOPC 21) was used (original magnification x 50). with the control myeloma antibody (Figure IC). Pattern 2 was charactcrized by the presence of many lymphoid cells (Figure 2A), but germinal centers were absent. This pattern was observed in 8 of 15 patients. Lymphoid cells in pattern 2 were predominantly Leu-4' T cells (Figure 2D), although R532' B cells (Figure 2B) were scattered throughout the sections in 4 (50%) of the 8 biopsy specimens. The absence of clusters of B532- cells was consistent with the absence of germinal centers on routine hematoxylin and eosin stains. When control myeloma protein MOPC 21 (Figure 2C) was used, only rarely were positive cells (less than 2%) seen in some biopsy specimens. Although the basis for this occasional false-positive staining remains unknown, these results emphasize the need to use control myeloma proteins of the Same class and concentration as the specific monoclonal antibodies being Pattern 3 showed fibroblastic proliferation with few (less than 50 per high power lymphoid cells (Figure 3A). The majority of the mono- SYNOVIAL MEMBRANE LYMPHOCYTES IN RA 35 lymphocytes reacted with this antibody. Synovial tissue obtained from osteoarthritic joints or from normal joints with traumatic injury showed relatively few lymphocytes, no proliferation of synovial and subsynovial fibroblasts, and little vascular proliferation, when routine histologic stains were used. Using monoclonal antibodies, approximately half the synovial lining cells were stained by anti-HLA-DR and HLADS antibodies; the subsynovial fibroblasts were not reactive. Only rare Leu-4' T cells or B532' B cells were present, and staining with anti-transferrin receptor or with anti-epidermal growth factor receptor was much less frequent than in the RA synovium. Cytofluorometric analysis. To more accurately quantitate the proportion of mononuclear cells in RA synovial membrane, cytofluorometric analysis was performed on cell suspensions eluted from synovial membranes by collagenase digestion. More T cells (56 Figure 3. A. Synovial membrane with pattern 3 infiltrate was stained with hematoxylin-eosin to show that only rare mononuclear cells were present. B and C, Staining with antibodies R532 and control myeloma MOPC-21. respectively. D, Reaction with antibody Leu-4. Arrow denotes positive staining. nuclear cells were Leu-4 ' T cells (arrow in Figure 3D). B532- B cells were rarely found (Figure 3B). All RA synovial membrane biopsy specimens demonstrated strongly positive staining with antiHLA-DR (antibody SC2) (Figures 4A and 5A). A similar pattern and intensity of staining of all synovial membrane biopsy specimens was noted with antiHLA-DS (antibody Leu-10) (Figures 4B and 5B). Transferrin receptors were demonstrated by staining with antibody 1 2 2 . Subsynovial cells, which were reactive in all three histologic patterns (Figures 4C and SC), were more prominent in pattern 3. Epidermal growth factor receptor (detected by antibody 455) was present on the vascular endothelial cells (Figures 4D and 5D); weaker staining was noted in the subsynovia] fibroblasts of some patients. In all 3 patterns, only rare (less than 20/o)were reactive with antibody F45 that detects the natural killer subset; this Was true even in patients in whom up to 40% of peripheral blood Figure 4. Pattern I rheumatoid arthritis synovial membrane was stained with: A, Monoclonal antibody SC2 (anti-HLA-DK); B. Antibody I.eu-lO (anti-HLA-DS); C. Antibody L22 (anti-transferrin receptor): D, Antibody 455 (anti+pidermal growth factor receptor). The space in the central part of the figure is the synovial cavity. Arrow denotes positive staining. YOUNG ET AL 36 * 12% Leu 4’ cells) than B cells (8 7% I3532 ’ or 6 2 2% IgD’ cells) were present. Lymphocytes stained with F45 comprised less than 3%, detected by cytofluorometry. A high frequency of HLA-DR+ cells (47 ? 12%) was present. Very few (<lo%) transferrin receptor cells were present. This is consistent with the infrequent staining of mononuclear cells on synovial tissue sections (Figures 4C and 5C), even though subsynovial fibroblasts were stained in these tissues. This selective expression of activation antigens has previously been noted on synovial fluid T cells (7). We found a positive correlation between the two methods (immunoperoxidase stained sections and cytofluorographic analysis of cell suspensions) for enumeration of the proportions of phenotypically distinct mononuclear cells. Analysis of peripheral blood lymphocytes (PBL) from these patients demonstrated a predom2 Figure 5. Pattern 3 rheumatoid arthritis synovial membrane was stained with the same panel of antibodies as used in Figure 4 including: A , Anti-HLA-DR; B, Anti-11I.A-DS; C, Anti-[ransferrin receptor; D, Anti-epidermal growth f x t o r receptor. Arrow denotes positive staining. inance of T cells (50-75% Leu-4’ cells) in all cases. In contrast to our prior study in which untreated RA patients’ PBL had a predominance of Leu-3a+ cells (ratio of Lcu-3dLeu-2a reactive cells = 2.4 2 0.3) (7), the previously treated KA patients in this study showed a wide variation in the proportion of their lymphocyte subsets (ratio of Leu-3dLeu-2a reactive cells = 0.9-2.4), There was no apparent correlation between pattern of synovial membrane infiltrate and ratio of subsets in their PBL. In 2 patients with pattern 3 infiltrate, synovial fluid lymphocytes were predominantly T cells, with ratios of Leu-3dLeu-2a reactive cells equaling I . 1 and 1.7, respectively; a high number of Ia+ T cells (21-40%) was present in each case. Clinical features. KA patients with pattern 1 were older at onset of symptoms (U < 0.05, Student’s t-test) and the duration of their symptoms before joint surgery was shorter than that of patients with patterns 2 or 3 (Table 1). All patients with pattern 3 had been treated with remittive agents, while 1 patient with pattern 1, and 2 patients with pattern 2 had not received these drugs. Continued active synovitis was present in patients with each pattern based on physical examination of the joints, radiographic evidence of progressive erosive joint destruction, and recurrent synovial effusions with high white blood cell counts and protein content. In particular, our 4 patients with pattern 3 did not appear to represent “burned out” inactive disease. Patients with pattern 3 were mildly anemic (hemoglobin = 12.3 5 0.7), 75% had erythrocyte sedimentation rate of greater than 30 mm/hour (mean 66 t 48 mm/hour), and all had rheumatoid factor titer greater than 1:640 (Table 1). Evaluation of hematoxylin-eosin stained synovial membranes. Since architecture of tissue is bett.er preserved in paraffin-embedded sections stained with hematoxylin-eosin, we compared results of assays on frozen tissue with those using routine histologic stains. We found that germinal centers (detected by antibody B532’ I3 cells) were more easily seen in the frozen tissue sections, especially when such germinal centers were small. The patterns found on frozen sections were in close agreement with those noted on the paraffin-embedded sections. Comparison of synovial samples obtained from different joints of the same individuals over periods up to 4 years demonstrated similar patterns of mononuclear cell infiltration in each patient. The range of joints biopsied for our immunohistologic studies is summarized in Table 2 and demonstrates that each pattern can be seen in any of the joints biopsied. SYNOVIAL MEMBRANE LYMPHOCYTES IN RA Table 2. Range of joints biopsied and synovial immunohistologic patterns observed Pattern Number of patients Number of joints 1 3 3 2 8 8 3 4 5 15 16 Total Joints biopsied 1 knee 2 wrists I hip 3 knees 4 wrists 1 hip 2 knees* 2 wrists * Two synovial membrane biopsies obtained 16 months apart from the left knee of I patient, the first at arthroscopic biopsy and the second at replacement arthroplasty. Synovial membrane biopsies from the 4 other knees and 2 hips were obtained at replacement arthroplasty and from the 8 wrists at synovectomy. DISCUSSION Three patterns of lymphoid cell infiltration of rheumatoid synovial membrane are described. These are characterized by abundant lymphoid infiltrates with germinal centers (pattern l), abundant lymphoid infiltrates without germinal centers (pattern 2), and fibroblastic/synovial proliferation with few lymphoid cells (pattern 3). Recent studies have emphasized synovial membrane biopsy specimens with heavy mononuclear cell infiltrates in order to characterize the cell-cell interactions that may occur. Previous studies by Fassbender and colleagues (reviewed in reference 23) have used routine histochemical stains to demonstrate the relative paucity of mononuclear cells in biopsy specimens obtained from some patients with active synovitis. Our results extend these observations by demonstrating a high frequency of dcndritic typc cells (HLA-DR+ , HLA-DS’, nonspecific esterasc negative) in biopsy specimens lacking lymphocytic infiltrates. Further, a high frequency of cells bearing transferrin receptor (TR) or epidermal growth factor receptor (EGFR) were found in these specimens. Such antigens provide markers for synoviocyte and fibroblast proliferation that occurs in the absence of direct T cell-synoviocyte contact and that may result from the action of soluble factors (24,25). The presence of particular cell types in synovial membrane that are not present in blood (such as B532 B cells, HLA-DR+/DS+ dendritic cells, and cells bearing EGFR and TR) are potentially important since such cells provide possible targets for selective drug therapy or immunoregulation. B532+ B cells are not + 37 specific for RA synovium since they also may be found in normal lymph nodes or in the inflammatory infiltrates in primary Sjogren’s syndrome (8). Conversely, few natural killer-like cells (< 3% antibody F45 reactive cells) were present in synovial membrane, although up to 40% of PBL were reactive. Taken together with previous studies on lymphocyte subsets in RA patients (7,16,26-29), the present study suggests the importance of “homing” of lymphocyte subpopulations (30-32) to different subregions of synovium or synovial fluid. Several different mechanisms may be responsible for the presence of distinct patterns of synovial membrane infiltration. A single patient may progress through different patterns, such as the evolution from pattern 1 through pattern 2 to pattern 3. The rate of progression through these various phases would depend on particular genetic and environmental factors, including medications. An alternative hypothesis is that distinct patterns exist in patients with early untreated disease and that such patterns persist throughout the disease course. Further studies in animal models of RA and prospective studies of untreated RA patients will be necessary to resolve these questions. In summary, we have found different patterns of synovial membrane infiltration in patients with active progressive RA. These results are in agreement with a preliminary report by Malone et al (33). These different patterns of infiltration may provide prognostic information or help predict responses to particular therapies in a manner analogous to the use of renal biopsy for patients with glomerulonephritis. However, long-term clinical followup using standardized treatment protocols will be necessary to evaluate the potential value of these measurements in rheumatologic practice. ACKNOWLEDGMENTS We wish to thank Ms Darla Harlow, Robert Buhrow, and David Finney for their excellent technical assistance. We are grateful to the members of the Divisions of Orthopedic Surgery (Drs. C. Colwell, R. Thorne, M . Hamer, and P. Hirshman) and Rheumatology (Drs. C. A. Robinson, G . W. Williams, P. K . Hench, J. G. Curd, and E. Tan) for allowing us to study their patients and Ms Shari Brewster for her secretarial assistance. Finally, we particularly wish to thank our Department of Pathology (Drs. J. Robb, G. Bordin, and W. S. Nichols) for their help in obtaining samples and the interpretation of pathologic Specimens and Dr. Frank Kozin for helpful discussions throughout these studies. We thank Dr. John Mendelson and Dennis Frisman (University of 38 YOUNG ET AL California, San Diego) for generous gifts of antibodies 455 and €3532, respectively. REFERENCES 1. Janossy G, Duke 0, Poulter LW, Panayi G, Bofill M , Goldstein G: Rheumatoid arthritis: a disease of T lymphocyte/macrophage immunoregulation. Lancet ii:839842. 1981 2. Duke 0, Panayi GS, Janossy G, Poulter LW: Immunohistological analysis of the lymphocytic infiltrates of rheumatoid synovial membrane using monoclonal antibodies. Ann Rheum Dis 41:192-193, 1982 3. Forre 0, 'I'hoen J , Lea T , Dobloug JH, Mellbye OJ, Natvig JB, Pahle J, Solheim BG: In situ characterization of mononuclear cells in rheumatoid tissues, using monoclonal antibodies: no reduction of T8-positive cells or augmentation in T4-positive cells. Scand J Immunol 16~315-319, 1982 4. 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Heidelberg, Springer, 1980, pp 2-143 24. Krane S: Aspects of the cell biology of the rheumatoid synovial lesion (The Heberden Oration). Ann Rheum Dis 40:433-488, 1981 25. Castor C, Roberts I),Hossler P, Bignal M: Connective tissue activation. XXV. Regulation of proteoglycan synthesis in human synovial cells. Arthritis Rheum 26522527, 1983 26. Konttinen Y , Keitamo S, Kanki A, Hayry P, Kankaanapaa U, Wegelius 0: Characterization of immunocompetent cells of rheumatoid synovium from tissue sections and eluates. Arthritis Kheum 24:71-79, 1981 SYNOVIAL MEMBRANE LYMPHOCYTES IN RA 27. Burmester G , Yu D, Irani A, Kunkel H , Winchester R: l a ' 'r cells in synovial fluid and tissues of patients with rheumatoid arthritis. Arthritis Rheum 24: 1370-1376, 1981 28. Klareskog L , Forsum U , Scheynius A, Kabelitz D, Wigzell H: Evidence in support of a self perpetuating HLA-DK dependent delayed type cell reaction in rheumatoid arthritis. Proc Natl Acad Sci USA 29:3632-3636, 1982 29. Duclos M, Zeidler H, Liman W, Pickier W, Rieber P, Peter H: Characterization of blood and synovial lymphocytes with rheumatoid arthritis and other joint diseases by monoclonal antibodies ( O K I ) series and acid napthyl esterase staining. Rheum Int 2:75-82 39 30. Kowley D, Gowans J, Atkins R, Ford W, Smith M: The specific selection and recruitment of recirculating lymphocytes by antigen in normals and pieimmunized rats. J Exp Med 137:499, 1972 31. Sprent J , Miller J , Mitchell G: Antigen induced selective recruitment of circulating lymphocytes. Cell Immunol 2:171. 1971 32. Van Boxel J, Paget S: Predominantly T cell infiltrate in rheumatoid synovial membranes. N Engl J Med 293: 5 17-520, 1975 33. Malone D. Cattell H, Tsokos M , Decker J. Wilder R: Immunohistologic heterogeneity of synovium from patients with clinically active rheumatoid arthritis (abstract). Arthritis Rheum (suppl) 26:S17, 1983 The Second Carl M. Peanon Memorial Symposium: Frontiers of Rheumatology March 15-17, 1984, Rancho Mirage, CA. Sponsored by the Annenberg Center for Health Sciences in association with the UCLA School of Medicine, Division of Rheumatology and the Southern California chapter of the Arthritis Foundation. CME credit available. Registration fee is $330/MDs; $180/fellows, trainees, and other health professionals. For additional information, contact Annenberg Center, for Health Sciences, Eisenhower Medical Center, 39000 Bob Hope Drive, Rancho Mirage, CA 92270, 800-321-3690 (toll-free, national), 800621-7322 (toll-free, California).