Excessive function of peripheral blood neutrophils from patients with behcet's disease and from hla-b51 transgenic mice.код для вставкиСкачать
ARTHRITIS & RHEUMATISM Volume 38 Number 3, March 1995, pp 426-433 0 1995, American College of Rheumatology 426 EXCESSIVE FUNCTION OF PERIPHERAL BLOOD NEUTROPHILS FROM PATIENTS WITH BEHCET’S DISEASE AND FROM HLA-B51 TRANSGENIC MICE MITSUHIRO TAKENO, A1 KARIYONE, NAOMI YAMASHITA, MASAFUMI TAKIGUCHI, YUTAKA MIZUSHIMA, HIDETOSHI KANEOKA, and TSUYOSHI SAKANE Objective. To elucidate the role played by HLAB51 in the neutrophil hyperfunction of Behqet’s disease, we determined the superoxide production by purified peripheral blood neutrophils from Behqet’s disease patients, from HLA-B51 positive healthy individuals, and from HLA-B51 transgenic mice, Methods. Neutrophil function was evaluated by flow cytometric analysis, detecting the conversion of 2’,7’-dichlorofluorescin diacetate into dichlorofluorescein, induced by superoxide in the neutrophils. Results. A significant correlation between the neutrophil hyperfunction and the possession of HLAB51 phenotype, regardless of the presence of the disease, was observed in humans. FMLP-stimulatedneutrophils (without in vitro priming) from HLA-BS1 transgenic mice, but not those from HLA-B35 transgenic mice or from nontransgenicmice, produced substantial amounts of superoxide. Conclusion. The HLA-BS1 molecule itself may be responsible, at least in part, for neutrophil hyperfunction in Behqet’s disease. Presented in part at the 57th Annual Scientific Meeting of the American College of Rheumatology, San Antonio, TX, November 10, 1993. Supported in part by research grants from the BehGet’s Disease Research Committee of Japan, Ministry of Health and Welfare of Japan; the Uehara Memorial Foundation, Tokyo, Japan; the Kanagawa Nanbyo Foundation, Kawasaki, Japan; and the Japan Intractable Diseases Research Foundation, Tokyo, Japan. Mitsuhiro Takeno, MD, PhD, Naomi Yamashita, MD, PhD, Yutaka Mizushima, MD, PhD, Hidetoshi Kaneoka, MD, PhD, Tsuyoshi Sakane, MD, PhD: St. Marianna University School of Medicine, Kawasaki, Japan; Ai Kariyone, PhD, Masafumi Takiguchi, MD, PhD: University of Tokyo, Tokyo, Japan. Address reprint requests to Tsuyoshi Sakane, MD, PhD, Institute of Medical Science, St. Marianna University School of Medicine, 2-16-1, Sugao, Miyamae-ku, Kawasaki, Kanagawa 216, Japan. Submitted for publication April 13, 1994; accepted in revised form October 5, 1994. Behget’s disease is a systemic inflammatory disease characterized by recurrent signs and symptoms of oral aphthosis, genital ulcers, skin lesions, and ocular involvement including uveitis. Aseptic neutrophi1 infiltration in the lesions is one of the most striking pathologic features, and various in vitro tests of neutrophil function in patients with BehGet’s disease have been shown to be abnormal (1-5). This suggests that the excessive neutrophil function plays a vital role in the development and clinical course of the disease. Epidemiologic studies have demonstrated that the disease is prevalent along the Silk Road: the Far East of Asia, China, the Middle East, and along the Eastern Mediterranean. These studies have also revealed that the HLA-B51 phenotype is strongly associated with Behget’s disease in these ethnic groups (6-10). Chajek-Shaul and coworkers have reported that neutrophil chemotaxis in patients with Behget’s disease who possess the HLA-B51 antigen was significantly enhanced as compared with that in HLA-B51 negative patients (4).Furthermore, Sensi and coworkers have recently reported that HLA-B51 could exert regulatory control on neutrophil functions (5). These results suggested that the HLA-B51 gene is one of the major genetic markers of neutrophil abnormalities in the disease. However, it remains to be determined whether the HLA-B51 molecule itself or its linked gene products are involved in the neutrophil hyperfunction. To elucidate the relationship between the hyperfunction of neutrophils and HLA-B51, we first determined the level of superoxide produced by purified peripheral blood neutrophils, avoiding the in vitro influences of immunocompetent lymphocytes and HLA phenotypes, in patients and in healthy subjects. We found that neutrophils from the patients produced significantly higher amounts of superoxide than did NEUTROPHIL FUNCTION IN BEHCET’S DISEASE those from the healthy controls. Moreover, the higher productivity of superoxide was closely related t o the possession of HLA-BS I , regardless of Behqet’s disease. These results suggest that the abnormal hyperfunction of neutrophils in patients with Behqet’s disease is determined in part by the HLA-BSI molecule. These observations prompted us t o generate HLA-BS 1 transgenic (Tg) mice t o examine the direct involvement of HLA-BSI in in vivo and in vitro functions of neutrophils. Although active lesions mimicking Behqet’s disease were not observed in any HLA-B51 Tg mouse, neutrophils from HLA-BS1 Tg mice responded t o FMLP without additional stimuli, and produced excessive amounts of superoxide; however, neither the HLA-B35 Tg nor the non-Tg mice showed this response. These findings suggest that the HLA-BS 1 molecule itself affects neutrophil function, which may play pivotal roles in pathogenesis and clinical course of Behqet’s disease. 427 A L. 01 . 1’0’ v PATIENTS AND METHODS Patients and controls. Twenty-nine Japanese patients with BehCet’s disease (16 male and 13 female), 14 of whom were HLA-B5I positive and 15 were B51 negative, were evaluated. The diagnosis was made based on the criteria proposed by the BehCet’s Disease Research Committee of Japan (1 I ) . Twenty-four age- and sex-matched healthy Japanese individuals, 9 of whom were HLA-1351 positive and 15 B51 negative, served as controls. HLA phenotypes were determined using a standard complement-dependent microlymphocytotoxicity test with internationally recognized antisera. Preparation of human peripheral blood neutrophils. Whole blood from patients and healthy donors was drawn into tubes containing heparin. Peripheral blood neutrophils were separated after centrifugation, at the interface between 2 Ficoll gradients of specific gravities 1.077 and 1 . 1 19. The purity of the neutrophils, determined by flow cytometric scattergram, was typically more than 97%. Measurement of superoxide production by human peripheral blood neutrophils. We measured neutrophil superoxide production according to the method described by Bass and coworkers (12), with slight modification. Briefly, purified neutrophils were gently diluted in Ca2’ -free and Mg2-‘free phosphate buffered saline (PBS) in a polypropylene tube (Corning, Corning, NY), and incubated with 5 pM 2‘,7‘dichlorofluorescin diacetate (Eastman Kodak, Rochester, NY) for 15 minutes at 37°C. After centrifugation, the neutrophils were resuspended in PBS, and an additional 15minute incubation at 37°C was performed in the presence of 1 pglml FMLP (Sigma, St. Louis, MO) or 100 nglml phorbol myristate acetate (PMA; Wako Pure Chemicals, Osaka, Japan). Cells generating superoxide in their cytoplasm became fluorescent from the conversion of 2‘,7’-dichlorofluo- Log Fluorescence Intensity Figure 1. Representative flow cytometric profiles of superoxideproducing neutrophils in A, unstimulated and B, FMLP-stimulated (1 pglml FMLP for 20 minutes) purified neutrophils. Superoxideproducing fluorescent neutrophils were determined by flow cytometry. A discriminating line was drawn at the point where <2% of unstimulated neutrophils could be assessed as positive. After FMLP stimulation, neutrophils that showed a shift to the right were considered positive (hatched area). rescin diacetate into dichlorofluorescein. Contaminating red blood cells were lysed with 0.83% ammonium chloride. The cytoplasmic content of superoxide in individual cells correlates with the intensity of fluorescence. Flow cytometric analysis was performed utilizing a flow cytometer (Cytron Absolute; Ortho, Rantan, NJ). The percentage of superoxide-producing neutrophils was calculated as the number of fluorescence-positive cells divided by the total number of cells examined. Figure 1 shows a representative flow cytometric profile of superoxide-producing neutrophils. Mice. Inbred C3H/He mice were purchased from Charles River Japan (Tokyo, Japan). HLA-B51 Tg C3H/He mice bearing the 6.4-kilobase Eco RI fragment of the HLAB*5101 heavy chain gene from a healthy Japanese individual were produced and characterized as previously described (13). HLA-B35 Tg mice bearing HLA-B*3501 were also 428 TAKEN0 ET AL A u molecules. Similar results were obtained in B35 Tg mice, but not in non-Tg mice (Figure 2). The magnitudes of mean fluorescence intensity of flow cytometric analyses were 8.23, 14.46, and 2.91 in lymphocytes and 10.35, 13.56, and 6.52 in neutrophils from B51 Tg mice, B35 Tg mice, and non-Tg mice, respectively. The specificity in HLA transgenic mice was determined by a complement-dependent cytotoxicity test using HLA-B51 and HLA-B35 antisera obtained from multiparous women (data not shown). Measurement of superoxide production by peripheral blood neutrophils from Tg and non-Tg mice. We measured superoxide production by murine neutrophils, utilizing the same method as used for humans, except that several stimuli, including 1 pg/ml FMLP, 100 nglml PMA, and 100 pdml opsonized zymosan (Sigma), were used. Whole blood white cells rather than purified neutrophils were used because of the technical difficulties in obtaining purified peripheral neutrophils from the limited amount of white cells 1 I from a single mouse. Flow cytometric analyses were performed on neutrophils by gating out lymphocytes and monocytes from neutrophils. The magnitudes of stimulant-induced superoxide production were evaluated by determining the relative fluorescence intensity ratio, calculated as the mean fluorescence intensity of stimulated neutrophrls divided by that of unstimulated neutrophils. Clinical and histologic examination. The existence of the clinical signs and symptoms typically seen in humans with BehGet's disease-skin lesions, oral aphthae, genital ulcers, and ocular lesions-was carefully documented ip the HLA-B Tg mice. Histologic studies were also performktl on those tissues and organs. Statistical evaluation. Statistical significance was examined using the unpaired Student's t-test. Log Fluorescence Intensity Figure 2. Expression of HLA-B transgene products on peripheral blood lymphocytes (A) and neutrophils (B)from HLA-B transgenic (Tg) mice. Peripheral blood cells were stained with fluorescein isothiocyanate-conjugated w6/32 monoclonal antibody specific for HLA class I molecules. After lysis of red blood cells, flow cytometric analysis was performed, with gating forward scatter and side scatter. Profiles of positive cells from an HLA-B51 Tg mouse (-), a B35 Tg mouse (- - -), and a non-Tg mouse (. * are a) shown. generated following the same procedures. The characterization of HLA-B35 Tg mice will be described in detail elsewhere (Kariyone A et al: manuscript in preparation). All Tg and non-Tg mice were bred and maintained under the same conditions. B51 Tg, B35 Tg, and non-Tg C3H/He mice were killed at ages 7-12 weeks for the in vitro neutrophil function tests. To determine the expression of HLA-B transgene products in Tg mice, peripheral lymphocytes and neutrophils were stained with fluorescein isothiocyanate-conjugated w6/32 monoclonal antibody, which has monomorphic specificity for the conformational elpitope of HLA class I molecules, and analyzed utilizing a flow cytometer. Figure 2 shows that peripheral lymphocytes as well as neutrophils from B51 Tg mice expressed the HLA class I heavy chain RESULTS Superoxide production by purified peripheral blood neutrophils from patients with Behget's disease. Highly purified peripheral blood neutrophils were stimulated with 1 pg/ml FMLP. The percentages of superoxide-producing neutrophils were calculated as described in Patients and Methods. The level of superoxide production by neutrophils from the patients was significantly higher than that from controls (mean ? SEM 53.8 ? 5.6% and 37.0 ? 5.9%, respectively; P = 0.046). The same data were analyzed according to the presence or absence of HLA-B51. Neutrophils from positive individuals, regardless of the presence of Behset's disease, produced much higher levels of superoxide than did those from B51 negative individuals, and the statistical significance of the difference was higher (mean 2 SEM 67.3 t 6.8% and 41.2 ? 10.6% in B51 positive and B51 negative patients [n = 291, P = 0.017; 52.6 ? 12.2% and 25.5 ? 4.7%, respectively, in controls [n = 241 P = 0.021; and 63.0 NEUTROPHIL FUNCTION IN BEHCET'S DISEASE " ( N = 1 I ) (N=9) (N=15)(N=14) controls patients ( N S O ) (N=23) combined Figure 3. FMLP-induced superoxide production by peripheral blood neutrophils from patients with BehGet's disease and from healthy controls. Superoxide-producing neutrophils from HLA-BS I negative and HLA-BSI positive subjects were determined by flow cytometry. Values are the mean and SEM 96 positive for superoxide. 429 ? 6.0% and 33.3 5 4.6%, respectively, among all study subjects [n = 531, P < O.OOOl), (Figure 3). When we compared superoxide production between B51 positive patients and B51 positive healthy individuals, the difference did not reach to statistical significance ( P = 0.263). Similar results were obtained for B5I negative patients versus B51 negative healthy individuals ( P = 0.09). PM A-induced superoxide production by purified peripheral blood neutrophils from the patients and controls was also evaluated. There were no statistically significant differences among these populations (mean t SEM 90.0 t 14.0%, 75.0 f 8.3%, 92.7 ? 17.2%, and 82.2 t 5.04% in B51 positive patients, B51 negative patients, B51 positive healthy individuals, and B51 negative healthy individuals, respectively). These results. thus suggested that the HLA-B5 1 antigen could regulate FMLP-induced neutrophil hyperfunction. Superoxide production by peripheral blood neutrophils from HLA-B Tg imice. To clarify the direct involvement of the HLA-lB51 molecule in neutrophil hyperfunction, we generaced HLA-B Tg mice. We examined superoxide production by peripheral blood B51 Tg B35 Tg non-Tg Log Fluorescence Intensity Figure 4. Representative profiles of superoxide production by FMLP-stimulated ( 1 pg/ml for I S minutes) neutrophils from 3 HLA-BSI transgenic (Tg) (A-C), 3 HLA-B3S Tg (D-F), and 3 non-Tg ( G I ) mice. Neutrophil fluorescence was assessed by flow cytometry of stimulated (-) and unstimulated (. . . .) neutrophils. 430 TAKEN0 ET AL PMA zymosan A B C D Log Fluoreseeme Intensity Figure 5. Representative profile,s of superoxide production by phorbol myristate acetate (PMA:. 100 ng/ml)-stimulated or opsonized zymosan (100 &ml )-stimulated neutrophils from HLA-B51 transgenic (Tg) (A and B), HLA-It335 Tg (C and D), and non-Tg (E and F) mice. Neutrophil fluorescence was determined by flow cytometry of stimulated 1-( and unstimulated (. . neutrophils. 9 0 ) neutrophils from the Tg mice induced by FMLP, PMA, or opsonized zymosatn to investigate the in vitro function of the neutrophils. Prominent changes were observed when the neutrophils were stimulated with FMLP. In B35 Tg mice and non-Tg mice, the magnitude of superoxide production by FMLP-stimulated neutrophils was comparable with that by unstimulated neutrophils (Figure 4). In contrast, neutrophils from B51 Tg mice responded vigorously to FMLP, producing high levels of superoxide without other stimuli such as lipopolysaccharide or cytokines (Figure 4). In contrast, neutrophils from not only the B51 Tg mice, but also the B35 Tg and non-Tg mice, responded well to PMA (Figures 5A, C , and E) and to opsonized zymosan (Figures 5B, D, and E), resulting in high levels of superoxide production. Figure 6 summarizes the relative fluorescence intensity ratios of the stimulated neutrophils compared with the unstimulated neutrophils from B51 Tg, B35 Tg, and non-Tg mice. The ratios in FMLP-stimulated neutrophils from B5 1 Tg mice were significantly higher than those in B35 Tg mice and those in normal non-Tg mice (Figure 6A). The mean ? SEM ratios were 1.53 2 0.09, 1.07 2 0.02, and 1.11 2 0.04 in B51 Tg mice, B35 Tg mice, and non-Tg mice, respectively (B51 Tg mice versus B35 Tg mice P < 0.001; B51 Tg mice versus non-Tg mice P < 0.001; and B35 Tg mice versus non-Tg mice P = 0.488). However, there were no differences in the distribution of the ratios of PMA-stimulated or opsonized zymosan-stimulated neutrophils among these 3 mouse groups (Figures 6B and C). The ratios of PMA-stimulated neutrophils were 3.72 2 1.40, 3.60 2 0.47, and 2.66 k 0.25 in B51 Tg mice, B35 Tg mice, and non-Tg mice, respectively (B51 Tg mice versus B35 Tg mice P = 0.922; B51 Tg B. PMA A. n l L P C. zymosan 5.0 4.0 15 3.0 0 t e: 2.0 B51Tg B35Tg nonTg 1.o B5lTg B35Tg nonTg BSlTg BJ5Tg nonTg Figure 6. Relative fluorescence intensity ratio of stimulated (with FMLP [A], PMA [B], or zymosan [C]) neutrophils to unstimulated neutrophils from 13 HLA-B51 Tg, 12 HLA-B35 Tg, and 17 non-Tg mice. The ratio was calculated by dividing the mean fluorescence intensity of stimulated neutrophils by that of unstimulated ones. * = P < 0.01 versus other 2 groups, by Student’s t-test. Values are the mean and SEM. See Figures 4 and 5 for concentrations of stimuli and for definitions. NEUTROPHIL FUNCTION IN BEHCET’S DISEASE mice versus non-Tg mice P = 0.363; and B35 Tg mice versus non-Tg mice P = 0.097). The ratios of opsonized zymosan-stimulated neutrophils were 2.52 2 0.19,2.78 +- 0.23, and 2.63 +- 0.27 in B51 Tg mice, B35 Tg mice, and non-Tg mice, respectively (B51 Tg mice versus B35 Tg mice P = 0.404; B51 Tg mice versus non-Tg mice P = 0.793; and B35 Tg mice versus non-Tg mice P = 0.727). We failed to demonstrate any correlation between responsiveness to the stimulants and differences in age or sex. These results indicated that peripheral blood neutrophils from B51 Tg mice were more sensitive to FMLP stimulation than were those from B35 Tg and non-Tg control mice. Clinical manifestations and histologic examination of mice. We investigated whether B51 Tg mice expressed clinical manifestations and pathologic changes up to 12 weeks of age. Neither clinical nor pathologic studies revealed any evidence of active lesions resembling those seen in humans with Behset’s disease (results not shown). DISCUSSION The significant association between Behset’s disease and HLA-B51 has been reported in several ethnic groups, especially among patients with ocular and/or neurologic involvement (610). It is believed that the principal biologic function of HLA molecules is to present antigens to the antigen-specific T cells, resulting in the triggering of the subsequent immunologic cascades. Therefore, the association suggests that T lymphocytes may be involved in the pathogenesis and/or the modification of the clinical course of the disease. One of us (TS) has reported the functional aberration of a suppressor T cell subset derived from patients with pre-active Behset’s disease (14). Kahan et a1 later reported a defect of the suppressive function in a CD4+ T cell subset (15). Recently accumulated evidence indicates that T lymphocytes bearing T cell receptor y/S (y/S T cells) are the major population in the infiltrating lymphocytes and their numbers are increased in peripheral blood (16,17). Their functional importance has also been reported from cloning studies of y/S T cells raised by activation with streptococcal antigens, and their pathogenetic importance in some patients with this disorder has been described (18). Among the streptococcal antigens, heat-shock proteins, especially the hsp65 family, might be one of the major epitopes for T cell lines and clones (19,20) 43 1 and might be responsible: for uveitis in susceptible rats (21). Neutrophil infiltration is an initial characteristic seen in skin and eye lesions of patients with BehFet’s disease, and the hyperfunction of peripheral blood neutrophils from such patients has been noted. Chajek-Shaul et a1 reported the elevation of neutrophil chemotaxis in HLA-BS 1 positive patients with Behset’s disease, and Sensi and coworkers showed enhanced neutrophil reactivity in HLA-B5 1 positive healthy individuals (43). Our data showing the positive association of the neiitrophil hyperfunction with the HLA-B51 phenotype. observed not only in humans but also in HLA-B Tg mice, directly demonstrate those previous observations. Neutrophil functions are regulated and maintained by inflammatory cytokines such as interleukin- 1 (IL-l), IL-2, IL-6, IL-8, tumor necrosis factor (TNF), granulocyte-macrophage colony-stimulating factor (GM-CSF), and granulocyte CSF (G-CSF). Among these cytokines, TNF ha:; been one of the primary interests in this field, because the structural genes of TNFa and TNFP are mapped within an HLA class I11 gene segment, and are in linkage disequilibrium with HLA class I genes (22). Our initial observations of cytokine production by purified neutrophils, but not by lymphocytes, suggest thlat not TNF production, but rather, IL-8 production rnight be regulated by the HLA molecule (unpublished observation). DeForge et a1 reported that mechanisms independent from TNF, IL-1, or IL-6 also could regulate IL-8 secretion, and that reactive oxygen metabolites regulate IL-8 production, suggesting circular or autocrine mechanisms between superoxide produci.ion and IL-8 (23). Taken together, our results suggest that the abnormal hyperfunction of peripheral blood neutrophils in the patients may be determined in part by an HLA-B51 molecule, and may occur in an autocirine manner through excessive production of proinflannmatory cytokines by those neutrophils. Several autoimmune diseases are closely associated with particular phenotypes of HLA. Generation of HLA transgenic animals is one of the useful strategies for establishing animal models for human diseases. For example, HLrbB27 Tg rats have been shown to have clinical features similar to those of human HLA-B27-related syndromes including ankylosing spondylitis (24). Wle generated HLA-B5 1 Tg mice as a candidate for an animal model to elucidate the contribution of HLA m,oleculesto the disease. We have shown here that FML,P-induced superoxide pro- 432 duction by neutrophils from HLA-B51 Tg mice is significantly elevated compared with B35 Tg or non-Tg mice. It is conceivable that the HLA molecules, which are xenogeneic to mice, could nonspecifically stimulate neutrophils in Tg mice. However, this is not the case, because this phenomenon occurred only in B51 Tg mice, and not in B35 Tg mice. The results suggest that the HLA-B5 1 transgene product itself is directly involved in neutrophil hyperactivity in these Tg mice. In the present study, only FMLP-induced superoxide production by neutrophils was increased in B51 Tg mice; PMA- or zyrnosan-induced superoxide production was comparable with that in other mice. The reason that neutrophills from B51 Tg mice are more sensitive to FMLP stimulation is not yet clear. Maximal superoxide production induced by FMLP requires “priming” by cytokines such as G-CSF, GM-CSF, and TNF (25). Our results suggest that neutrophils in B51 Tg mice may be easily agitated intrinsically to produce superoxide by stimuli that are not potent enough to cause neutrophils from B35 Tg or non-Tg mice to produce a large amount of superoxide. Alternatively, neutrophil hyperfunction observed in B51 Tg mice might represent an inherited abnormality in their immune systems, thereby allowing them to secrete excessive cytokines to prime neutrophils in vivo. Possession of the E:[LA-B51 molecule is not sufficient to induce clinical disease among the Tg mice. This is similar, however, to the relationship between the human disease and HLA-B51. Even in Japan, the area with the highest prevalence, Behset’s disease occurs in only 1 in 1,000 individuals who have the HLA-B5 1 phenotype (6). Although intrafamilial occurrences of Behset’s disease related to HLA-BS 1 have been observed (26,27:), the coincidence of Behset’s disease in monozygotiic twins is rarely reported. These data indicate that 13LA-BSl is not a single genetic factor which determines the occurrence of the disease. There is no HLA-B association in Caucasian patients with Behset’s disease. Lehner and colleagues emphasized the importance of heat-shock proteinresponsive lymphocytes for the onset of the disease in Caucasian patients (19), whereas we believe the infiltration of peripheral blood neutrophils is the primary phenomenon in Japanese patients with Behset’s disease. These facts might reflect that Behset’s disease is not a single clinical entity, but rather, a clinical syndrome (21). The phenotype in combination with environmental factors, such as bacterial (28) or viral infec- TAKEN0 ET AL tions (29), may lead to the development and/or exacerbation of the disease in humans. REFERENCES 1. Matsumura N, Mizushima Y: Leukocyte movement and colchicine treatment in BehGet’s disease. Lancet 2:813, 1975 2. 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Acta Med Scand 220:375-378, 1986 Arber N, Klein T, Meiner Z, Pars E, Weinberger A: BehGet’s syndrome: epidemiology, clinical data, and HLA typing in Israeli patients and their families, BehCet’s Disease. Edited by JD O’Duffy, E Kokmen. New York, Marcel Dekker, 1991 Mizushima Y: BehCet’s disease. Cum Opin Rheumatol3:32-35, 1991 Eglin RP, Lehner T: Detection of RNA complementary to herpes-simplex virus monoriuclear cells from patients with BehCet’s syndrome and recurrent oral ulcers. Lancet 2:13561360, 1982 New Manuscripts to be Sent to Dr. William P. Arend as of April 1 Dr. William P. Arend will officially assume the full responsibilities of Editor, Arthritis and Rheumatism, on July 1, 1995. However, as part of the transition from the Editorship of Dr. Peter H. Schur, Dr. Arend will handle the review process for all new manuscripts submitted on or afler April 1, 1995. Any manuscripts submitted on or afler that date should be sent to William P. Arend, MD, Editor, Arthritis and Rheumatism, University of Colorado Health Sciences Center, Box B-117, 4200 E. Ninth Avenue, Denver, CO 80262.