Hematologic and cytofluorographic analysis of patients with feltyaposs syndrome a hypothesis that a discrete event leads to large granular lymphocyte expansions in this condition.
код для вставкиСкачатьARTHRITIS & RHEUMATISM Vol. 38, No.9, September 1995, pp 1252-1259 0 1995, American College of Rheumatology 1252 HEMATOLOGIC AND CYTOFLUOROGRAPHIC ANALYSIS OF PATIENTS WITH FELTY’S SYNDROME A Hypothesis that a Discrete Event Leads to Large Granular Lymphocyte Expansions in this Condition S. J. BOWMAN, V. CORRIGALL, G. S. PANAYI, and J. S. LANCHBURY Objective. To compare hematologic and cytofluorographic features in Felty’s syndrome (FS) patients with and without the large granular lymphocyte (LGL) syndrome. Methods. Peripheral blood cells from FS patients and from 2 control groups (rheumatoid arthritis [RA] patients and subjects without symptoms of a rheumatic disease) were analyzed by hematologic and cytofluorographic techniques. A separate assessment of disease activity was performed. Results. FS patients had reduced lymphocyte and platelet counts, with a parallel reduction in lymphocyte subsets examined. CD4 counts were reduced in all FS patients, including those with the LGL syndrome. Disease activity was lower in FS patients than in RA control patients. Treatment was similar in all patient groups. No direct association was seen between LGL numbers and duration of RA or neutrophil counts in RA groups. Conclusion. Hematologic abnormalities in FS extend beyond neutropenia. Although similarities were seen between FS patients and FS patients with the LGL syndrome (e.g., CD4 lymphopenia), evidence for a gradation from FS to the LGL syndrome was not seen, thus favoring the hypothesis that a “transforming event” is required. Felty’s syndrome (FS), which comprises rheumatoid arthritis (RA), leukopenia, and often splenomegaly (l), occurs in -1% of RA patients. Between 15% and 40% of FS patients also have a form of T cell “leukemia,” the large granular lymphocyte (LGL) syndrome (2-4). In this latter syndrome, there is an expansion of peripheral blood T lymphocytes that have a large granular morphology. Conversely, a number of studies have estimated that up to 30% of patients with the T cell LGL syndrome have evidence of RA (5). LGLs comprise -15% of normal peripheral blood lymphocytes, although they increase in number with age (6). LGLs include natural killer (NK) cells as well as T cells, which are of interest in the present study. The most common surface phenotype of T cell LGLs in FS patients is CD3+,CD8+,CD57+, CD16+/-. CD56, a marker seen on NK cells, is less commonly expressed on T cell LGLs. This study was directed toward identifying hematologic features and lymphocyte subsets in patients with FS, with or without the LGL syndrome, in RA control patients, and in a group of control subjects without symptoms of rheumatic diseases, to give further insight into the pathogenesis of these conditions. PATIENTS AND METHODS Supported in part by the Arthritis and Rheumatism Council, UK. Dr. Bowman’s work was supported by an MRC Training Fellowship. S . J. Bowman, MRCP, V. Comgall, PhD, G. S. Panayi, FRCP, J. S. Lanchbury, PhD: UMDS, Guy’s Hospital, London, UK. Address reprint requests to J. S. Lanchbury, PhD, Division of Medicine, UMDS, Guy’s Hospital, London SE1 9RT, UK. Submittted for publication December 2, 1994;accepted in revised form March 20, 1995. Patient population. Fifty-five FS patients were recruited for this study. All fulfilled the American College of Rheumatology (formerly, the American Rheumatism Association) 1987 criteria for RA (7). In addition, they all had a current or previous history of an unexplained neutropenia (neutrophil count below 2 x 10’Aiter) that persisted for at least 6 months. Splenomegaly was not an essential criterion for the diagnosis of FS in this study, a guideline that is now generally accepted (1). All patients were Northern European 1253 HEMATOLOGY OF FS AND LGL SYNDROME Caucasoids except for 1 patient, who was of Turkish Cypriot origin. These patients are described in detail elsewhere (4). Briefly, 17 of them had evidence of the LGL syndrome, while 32 did not. The remaining 6 patients could not be categorized into either group and were excluded from further study. The LGL syndrome was defined as either an absolute lymphocytosis of LGLs above 1 X 10’hiter or an increased percentage of LGLs of at least 25% of the total lymphocytes, persisting for at least 6 months. LGLs were identified by morphology and immunophenotyping, and 15 of 17 had evidence of clonal disease based on restriction fragment length polymorphism (RFLP) or sequencing analysis (4). Control population. Two control groups were studied. Since the number of LGLs increases with age (6), it was important to ensure that subjects in the control groups had the same age range as those in the FS groups. The first control group was an outpatient group of 23 randomly recruited RA patients attending Guy’s Hospital outpatient clinics. The second was a group of individuals without rheumatic disease symptoms (non-rheumatic disease [NRD] controls). This comprised both hospital staff members and individuals attending outpatient clinics at Guy’s Hospital for followup of routine medical conditions such as hypertension. All were Northern European Caucasoids, except for 2 of the RA patients (1 Greek Cypriot, 1 Asian) and 1 of the NRD controls (Greek Cypriot). Cell separation, immunofluorescence, and flow cytometry. Venous peripheral blood was collected into tubes containing preservative-free heparin, and mononuclear cells were separated on a Lymphoprep (Nycomed, Oslo, Norway) density gradient. Immunophenotyping was performed by flow cytometry (fluorescence-activated cell sorter [FACS] analysis) using a Becton Dickinson FACScan (Oxford, UK). Double staining was performed using the following phycoerythrin- and fluorescein isothiocyanate (FITCt conjugated monoclonal antibodies: mouse IgGl antibodies as negative controls, anti-CD3, anti-CD4, anti-CD8, antiCD16, anti-CD56, anti-CD57, anti-T cell receptor alp and ylS, and FITC-conjugated goat anti-mouse antibodies (all from Becton Dickinson). Standard complete blood cell counts and white blood cell differential counts were used to calculate absolute numbers of each lymphocyte subset. Disease activity. The European League Against Rheumatism (EULAR) core data set (8) was used to assess RA activity in a proportion of the FS patients and RA controls. This comprised the number of swollen joints (28 joint count), number of tender joints (28 joint count), joint pain (10-cm visual analog scale [VAS]), patient’s global assessment of disease activity (10-cm VAS), duration of morning stiffness (minutes), and the erythrocyte sedimentation rate (ESR). In addition, a modified disease activity score (DAS) (9) was calculated according to the following formula: DAS = (0.555 x d n tender joints) + (0.284 x d n swollen joints) + (0.7 X In ESR) + (0.0142 x patient’s global assessment), where the maximum “n” value is 28. Statistical analysis. The Mann-Whitney U test and chi-square test were used to compare results between groups. P < 0.05 was considered significant. For correla- tions between variables, Spearman’s rank coefficient of correlation was used. RESULTS Clinical features. Table 1 shows the comparison of basic clinical data among the 4 groups. The salient features are as follows. The LGL group was older than the other groups and had developed neutropenia at an older age. The mean time period between the development of RA and the development of FS was 12 years in the FS group and 15 years in the LGL group, but with enormous variation from patient to patient. The female-to-male ratio in the FS and LGL patients was less than that in the RA patients. Thirteen of the 32 FS patients (41%) and 2 of the 17 LGL patients (12%) currently had a neutrophil count above 2 x 10’Aiter (both LGL patients had clonal LGL expansions by RFLP analysis [4]). Splenomegaly was more common in the FS patients than in the LGL patients. All patient groups had a high incidence of seropositivity for rheumatoid factor and erosive disease. Approximately one-third of the FS and LGL patients had a history of infections. Most of those whose bone marrow had been analyzed had an active marrow, with only 2 of 18 FS patients (11%) having decreased granulopoiesis. Four of 11 LGL patients (36%) and 3 of 18 FS patients (17%) had bone marrow lymphocytosis. Analysis of the peripheral blood from the 3 FS patients showed no evidence of the LGL syndrome. Drug treatment in the FS, LGL, and RA patient groups was similar. Complete blood cell count parameters. By definition, the FS and LGL groups had a significantly reduced neutrophil count compared with the control groups (Table 2). In the FS group, the lymphocyte and platelet counts were also reduced, which was not seen in the LGL group. The red blood cell count was not affected in either the FS or the LGL group. The RA patient controls also differed from the NRD controls. The neutrophil and platelet counts were significantly increased, and the hemoglobin reduced. This is all suggestive of active inflammatory disease. The mean ESR in these patients was also substantially increased at 47 mm/hour, with an ipterquartile range of 32-58 mm/hour, which would be compatible with active inflammation. Three RA patients were lymphopenic (lymphocyte count below l x 10’Aiter; interquartile range 0.83-0.98 X lO’Aiter), as was 1 individual from the NRD control group (0.91 X 1O’Aiter). BOWMAN ET AL 1254 Table 1. Clinical features of the study groups* RA patients NRD controls LGL patients (n = 17) (n = 23) (n = 20) FS patients (n = 32) ~ Age-Females:males, n (ratio) RA duration, mean f SD years Age at RA onset, mean f SD years FS duration, mean f SD years Age at FS onset, mean f SD years RA-FS period, mean t SD years Neutrophils now >2 x lO’Iliter, n (%) Splenomegaly, n (%) RF positive by latex test, n (%) Radiographic erosions, n (%) Infections, n (%) Bone marrow, dtotal tested Active Lymphocytosis Decreased granulocytes Current treatment, n Prednisolone f DMARD DMARD 2 NSAID NSAID alone 6 0 f 127 14:18 (0.8) 17 f 11 43 f 14 4 f 5 56 f 126 12 f 11 13 (4117 28/32 (88)# 28/32 (88) 23/19 (79) 9/32 (28) 63 f 11$ 17:6 (2.8) 15 -+ 8 48 & 13 NA NA NA 23 (100) NK 19/23 (83) 11/13 (85) NK 60 f 21 13:7 (1.9) NA NA NA NA NA 20 (100) NK NK NA NK 71 t 10 10:7 (1.4) 20 f 15 50 f 18 5 f 5 66 f 12 15 f 13 2 (12) 9/17 (53) 14/17 (82) 9/16 (56) 6/17 (35) 14/18 3/18 2/18 NK NK NK NK NK NK 11111 411 1 0111 11 9 12 8 9 6 NA NA NA 10 3 4 * P values determined by Mann-Whitney U test (for continuous data) or by chi-square with Yates’ correction (for discontinuous data). See Patients and Methods for definitions of the study groups. FS = Felty’s syndrome; RA = rheumatoid arthritis; NRD = non-rheumatic disease; LGL = large granular lymphocyte syndrome; NA = not applicable; NK = not known; RF = rheumatoid factor; DMARD = disease-modifying antirheumatic drug; NSAID = nonsteroidal antiinflammatory drug. t P = 0.004 versus LGL group. $ P = 0.03 versus LGL group. 1 P = 0.009 versus LGL group. ll P = 0.05 versus LGL group. # P = 0.02 versus LGL group. reduction in CD8 counts (0.22 ? 0.13 X lO’/liter), while in the LGL group it was increased (2.60 2.55 x lO’/liter). The RA group had a borderline reduction in CD8 cells (0.29 ? 0.23 x 109/liter)compared with the NRD control group (0.39 ? 0.24 x l@/liter) (P= 0.046). Examination of the CD4:CD8 ratio is instructive. In the LGL group, the ratio was grossly abnormal FACS analysis of peripheral blood lymphocyte phenotype. The CD3 count in the FS group (mean ? SD 0.67 0.36 x 10’hiter) was significantly reduced compared with the counts in the other groups (Table 3). The CD4 count was reduced in both the FS group (0.40 0.24 x 10’hiter) and the LGL group (0.41 ? 0.38 X lO’/liter). In the FS group, there was a similar * * * Table 2. Hematologic data on the study groups* Neutrophils, x 10’Iliter Lymphocytes, xlO9/Iiter Platelets, x lo’fliter Hemoglobin, g d d l FS patients (n = 32) RA patients (n = 23) NRD controls (n = 20) LGL patients (n = 17) 1.89 f 1.647 0.99 f 0.42$ 155 f 771 12.6 f 1.4 6.12 f 1.957 1.60 f 0.53 304 f 9571 12.1 f 1.2# 4.18 5 1.227 1.85 f 0.68 234 f 728 13.0 f 1.3 1.27 5 1.257 3.62 f 3.17 207 t 99 12.5 f 1.5 * The mean f SD erythrocyte sedimentation rate in the RA patients was 47 f 28 mdhour (range 5-130, interquartile range 32-58). Values are the mean f SD. See Table 1 and Patients and Methods for definitions. 7 All groups differed significantly from all other groups at P = 0.0002, except for the FS group versus the LGL group, which were not significantly different from each other. $ P = 0.0003 versus all other groups. 1P = 0.006 versus all other groups, except the LGL group. llP = 0.006 versus all other groups, including the LGL group. # P = 0.02 versus NRD controls. HEMATOLOGY OF FS AND LGL SYNDROME 1255 Table 3. Flow cytometry findings in the study groups* RA patients (n = 23) NRD controls (n = 20) LGL patients (n = 17) 1.06 f 0.35t 0.73 2 0.298 0.29 f 0.23# 3.6 f 2.3 0.24 f 0.20 20.9 2 14.1 0.013 f 0.018 0.070 -t 0.078## 1.31 f 0.66 0.84 f 0.439 0.39 f 0.24 2.5 f 1.3 0.24 f 0.16 19.2 f 13.3 0.021 f 0.023 0.117 2 0.074 3.25 -t 3.05 0.41 f 0.38 2.60 f 2.55** 0.3 & 0.3** 2.08 f 2.53** 57.0 f 20.9** 0.825 f 1.72289 0.163 f 0.360 FS patients (n = 32) 0.67 f 0.36t CD3 count, x 109/liter CD4 count, x lphiter 0.40 f 0.249 CD8 count, X 10’hiter 0.22 f 0.13ll CD4:CD8 2.2 f 1.4tt CD3+,CD57+, xl@hter 0.12 f 0.09$$ CD57 +/CD3 + , % 18.9 & 11.0 CD3+,CD16+, X 109/Iiter 0.017 f 0.032 CD3+,CD56+, X lO’/Iiter 0.030 f 0.032Tll * Values are the mean f SD. See Table 1 and Patients and Methods for definitions. t P = O.OOO4 versus all other groups. $ P = 0.014 versus LGL group. 9 P = O.ooO1-O.OOO8 for FS versus RA and NRD groups, for RA versus FS and LGL groups, and for NRD versus LGL group. ll P = 0.002 versus NRD group. # P = 0.046 versus NRD group. ** P = O.OOO1 versus all other groups. tt P = 0.008 versus RA group. S$ P = 0.027 versus RA group and P = 0.007 versus NRD group. 98 P = 0.013 versus FS group and P = 0.031 versus RA group. llllP = 0.001 versus RA and NRD groups. ## P = 0.01 versus NRD group. (mean 2 SD 0.3 _t 0.3), as expected. The ratio in the FS group (2.2 ? 1.4) was very similar to that in the NRD control group (2.5 ? 1.3), suggestive of a parallel reduction in CD4 and CD8 cells. In the RA patients (3.6 ? 2.3), the CD4:CD8 ratio was increased, although not significantly, compared with the NRD controls. By definition, CD3+,CD57+ counts were significantly increased in the LGL group compared with the other groups (2.08 -t 2.53 X 10’hiter; P = 0.0001). However, while they were reduced in the FS group compared with the levels in the RA and NRD control groups, the percentage expression was unchanged, again suggestive of a parallel decrease in all of the lymphocyte subtypes examined (Figure 1). CD16 showed an increased expression in the LGL group compared with the FS and RA groups, while CD56 expression was reduced in the FS and RA groups. With age, the absolute lymphocyte count decreases, while the percentage of T cells that express CD57 increases (6). One previous study of FS failed to specify the age of the control group (3). In the present study, NRD control samples came from both laboratory staff and outpatients undergoing routine followup for conditions such as hypertension, which is commonly present in older individuals. There were no significant differences between the 2 subgroups with 8 Figure 1. Comparison of hematologic and cytofluorographic features in 32 patients with Felty’s syndrome (FS) alone, by treatment group. Group 1, FS pred+ = 11 patients taking oral prednisolone (pred) with or without disease-modifying antirheumatic drugs (DMARDs) or nonsteroidal antiinflammatory drugs (NSAIDs). Group 2, FS DMARD = 9 patients taking DMARDs with or without NSAIDs. Group 3, FS NSAID = 12 patients taking NSAIDs or no antirheumatic treatment. * P = 0.025 group 2 versus group 3. The values for age, rheumatoid arthritis (RA) duration, and FS duration were divided by 10, and the platelet count by 100, in order to fit the scale of the figure. 1256 BOWMAN ET AL Table 4. Spearman correlation coefficients (r) for comparisons of CD57 expression as a percentage of CD3+ cells (%), and CD57+,CD3+ absolute cell counts (count), with the variables specified, in the study groups* RA patients r = 0.43t (n = 23) FS patients r = 0.35t (n = 32) % Age (years) RA duration (years) FS duration (years) Neutrophils (Xl@Aiter) 0.38 0.16 0.02 -0.07 0.14 0.09 0.14 LGL patients r = 0.51t (n = 17) % Count % Count % Count 0.01 0.14 NA -0.18 0.05 0.07 NA -0.04 0.56 NA NA 0.38 0.26 NA NA 0.36 -0.01 -0.25 -0.07 0.39 0.22 -0.13 -0.13 0.19 Count -0.25 NRD controls r = 0.45t (n = 20) * See Table 1 and Patients and Methods for definitions. t r value for P < 0.05, which is considered significant. regard to any of the parameters analyzed except for age (43.8 2 14.7 versus 76.3 t 10.8; P = 0.0004 by Mann-Whitney U test) and, consequently, CD3+, CD57+ expression (12.8 2 9.5% versus 25.6 +. 13.9%; P = 0.0284 by Mann-Whitney U test). Comparison between FS patients with current neutropenia and those with a neutrophil count currently above 2.0 x 109/liter. Comparison of the 19 FS patients currently neutropenic with the 13 who were no longer neutropenic found few significant differences. Clearly, the neutrophil counts were different (0.89 +. 0.45 versus 3.34 2 1.64 x 10g/liter; P = O.OOO1 by MannWhitney U test). The duration of FS was also signifi3.38 cantly different between these groups (3.00 versus 6.31 2 5.92 years; P = 0.048 by Mann-Whitney U test). The longer the duration of disease the more likely the neutrophil count has had time to recover. There were no significant differences between these two groups with regard to any of the other laboratory indices. There was a significant sex difference in the occurrence of neutropenia. Twelve of the 19 currently neutropenic patients (63%) and 2 of the 13 of those whose counts had recovered (15%) were males (P = 0.021 by chi-square test). Eighteen of the 19 currently neutropenic patients (95%) were HLA-DR4 positive, compared with 8 of the 13 non-neutropenic patients (62%) (P= 0.057). Treatment. One important issue with regard to FS patients is whether treatment could account for some or all of the abnormalities. We categorized the patients according to 3 main treatment groups: 1) patients taking oral steroids with or without other treatments, such as disease-modifying antirheumatic drugs (DMARDs) or nonsteroidal antiinflammatory drugs (NSAIDs). 2) Patients taking a DMARD, with or * without an NSAID, but not taking oral steroids. 3) Patients not taking oral steroids or a DMARD, but might be taking an NSAID. The only significant difference was that patients in treatment group 3 were older (67 2 9 years) than those in treatment group 2 (52 f 16 years) (P = 0.03 by Mann-Whitney U test) (Figure 1). Correlation of CD3+,CD57+ expression with other variables in patient and control groups. In the NRD control group, a correlation of increasing age with the percentage of CD3 cells that coexpressed CD57 was found (Spearman’s r = 0.56) (Table 4). Absolute numbers of CD3-t ,CD57+ cells did not show this correlation. There was a significant inverse correlation between age and total lymphocyte count (r = -0.55) in the NRD controls. The findings in the FS patients were similar to those in the NRD controls. However, in the RA and the LGL patient groups, this relationship was not seen. No correlation was seen between the percentage expression of CD57 on CD3 cells and disease duration in any of the patient groups (Table 4). No inverse relationship between the numbers of LGLs (as shown by CD3+,CD57+ cells) or CD57/CD3% expression and the peripheral blood neutrophil count was seen in any of the groups studied (Table 4). Disease activity. A comparison of disease activity between 17 of the 49 FS patients (35%; 10 with FS alone and 7 with FS and the LGL syndrome) and 14 of the 23 RA patients (61%) showed that all activity indices were lower in the FS patients than in the RA patients, although these differences did not all reach statistical significance (Table 5). The modified DAS was significantly lower in the FS group (P = 0.008). Numbers were insufficient to analyze the FS patients with and without the LGL HEMATOLOGY OF FS AND LGL SYNDROME Table 5. Comparison of disease activity measurements in 17 FS patients (10 with FS alone and 7 with FS and LGL syndrome) and 14 RA patients* stiffness, minutes Patient’s assessment of pain (10-cm VAS) Patient’s global assessment (10-cm VAS) No. of swollen joints (maximum 28) No. of tender joints (maximum 28) Erythrocyte sedimentation rate (mmhour) Modified Disease Activity Score AM FSLGL patients (n = 17) RA patients 44 f 62 3.0 f 1.8t 114 2 167 5.3 2 2.9 2.4 2 1.7$ 4.7 3.5 6.4 f 6.5 f 4.0 3.2 2 3.8 (n = 14) 7.8 2 k 2.7 8.7 34 2 38 39 2 24 2.9 f 1.25 4.5 f 1.4 * Values are the mean 2 SD. VAS = visual analog scale. See Table 1 and Patients and Methods for other definitions. = 0.037 versus RA group. = 0.020 versus RA group. 5 P = 0.008 versus RA group (P = 0.006 for 10 patients with FS alone versus RA group). tP tP syndrome as separate groups. A comparison between the 10 patients with FS alone and the 14 RA patients yielded similar results, but with more of the indices differing significantly (P = 0.006) (Table 5). DISCUSSION The term Felty’s syndrome is now used to describe the association of RA with neutropenia (1,lO). Although splenomegaly is common, it varies over time and in extent, and it may be absent (1,lO). We have provided evidence that hematologic abnormalities in FS extend beyond neutropenia, with parallel reductions in CD4 and CD8 cells. A detailed comparison of the hematologic features and T lymphocyte subsets in FS patients with and without LGL expansions, as well as in RA patients without neutropenia, has provided important clues to the relationship between these conditions. Most importantly, evidence of a gradation from FS to the LGL syndrome was not seen, thus favoring the hypothesis that a “transforming event” is required. The neutropenia in FS is variable. In some patients it is profound, in others it fluctuates, and in a proportion of patients it recovers completely (1,3,10). In the present study, patients in this latter group were more likely to be female and to have a lower frequency of HLA-DR4. As an explanation for this difference, we cannot exclude the theoretical possibility that HLA-DR4 positive male patients die early in the 1257 course of FS. The recovery may occur after a long period of neutropenia. One of the patients described in our study, for example, has a severe seropositive, erosive, destructive arthropathy (now quiescent), splenomegaly, and is positive for HLA-DR4. She had a profound neutropenia, with a nadir of 0.1 X lO’Aiter, over an 8-year period, which resolved spontaneously. Although neutropenia is the most prominent feature, FS is a more generalized disease that may affect other cell types. Anemia, lymphopenia, and thrombocytopenia have all been described (10) to various extents (1). It has been suggested that one reason for these findings is hypersplenism. In this study, however, the presence of splenomegaly was not related to the levels of these cell types in the blood, and the red blood cell count was undected, which is unusual for hypersplenism. Although it is clear that the incidence of infection in FS patients is, in general, related to the degree of neutropenia (1l), this is by no means absolute, and the low CD4 count may be an additional contributing factor. Patients with FS and LGL expansions also had low CD4 counts, and this may imply that there are further similarities in the pathogenic mechanisms of these 2 variants of FS. CD3+,CD16+ cells, and to a lesser extent CD3+,CD56+ cells, were higher in the LGL group, but there was enormous variation from patient to patient, since individual LGL expansions either expressed or did not express these markers. They were also reduced in parallel with the other lymphocyte subsets in the FS patients. The possibility that antiarthritic drugs could account for these variations was also examined (Figure 1). No difference was found between FS patients grouped with regard to antirheumatic treatment, except that DMARDs were less common in older patients. This may reflect a “bum-out” of the disease in the older patients, or it may mean that the physician prefers less-aggressive therapy in this group. It is clear, however, that oral steroid use cannot account for the abnormalities seen in the FS patients, and although DMARDs appear to be effective treatment for the neutropenia, there is little evidence that steroids are of any benefit (1). One hypothesis for the presence of LGL expansions in patients with FS is that they represent a spectrum of disease with a potential for progression over time, from FS to FS with the LGL syndrome. The FS group as a whole might have been expected to show some sign of this with a relative increase in CD3+,CD8+,CD57+ cells. This was not found. The FACS analysis demonstrated that FS patients had a 1258 parallel reduction in all of the lymphocyte subsets demonstrated. There was no relative preservation of CD3+,CD57+ cells to suggest a “low level” LGL syndrome. That is, rather than a complete gradation from FS to the LGL syndrome, a saltatory event seems to take place, transforming FS to the LGL syndrome. This could be required for clonal LGL expansion. This does not exclude the possibility, however, that functionally abnormal cells, whether clonal or not, are present in all FS patients but cannot be detected by current techniques. Further work, using more sensitive polymerase chain reaction-based techniques to assess T cell clonality, is required to address this question. The mechanisms leading to LGL expansion in patients with RA may differ from those in patients without RA. We have previously demonstrated that the prevalence of HLA-DR4 is increased in LGL syndrome patients with RA, but not in those without RA (12). It is of interest that in celiac disease (glutensensitive enteropathy), an immune-mediated disorder in which there is a close association with HLADQAl”O501;DQB1*0201, there is an increased risk of enteropathy-associated T cell lymphoma (EATCL) (13). Not all patients with EATCL have evidence of celiac disease, but in contrast to the LGL syndrome, EATCL arises in patients with the same HLA genotype that predisposes to celiac disease (13). No direct correlation between the levels of neutrophils and CD3+ ,CD57+ cells in the peripheral blood was seen in this study. Even if LGLs are involved in the peripheral suppression or destruction of neutrophils, it could be argued that a direct relationship between the 2 is too simplistic an expectation for a potentially complex biologic process. It is of interest that in RA patients, with active disease, a reduction in CD8 counts has been reported (14). In this study, a borderline reduction in CD8 counts was seen in EL4 patients, while the neutrophil and platelet counts were increased, which is the opposite of FS. FS could result from a down-regulation of the cytokine(s) putatively responsible for this (e.g., interleukin-1 [IL-l]/IL-6), secondary to chronic stimulation. One hypothesis is that a switch could occur from THl cells involved in stimulating cellular immunity (and active joint disease via IL-2 and/or interferon-y) to TH2 cells involved in humoral immunity (and hence, less active joint disease, and a higher incidence of B cell activity, e.g., rheumatoid factor positivity and antineutrophil antibodies via cytokines such as IL-4hL-5). Similar hypotheses have been proposed in rela- BOWMAN ET AL tion to the progression of human immunodeficiency virus infection to acquired immunodeficiency syndrome (15). In support of this hypothesis, there is evidence that although the severity of erosive joint disease in FS is similar to that in RA patients with similar disease duration (1,16), the disease activity (i.e., active synovitis) is lower in FS patients (16). The erosions may reflect the outcome of earlier (i.e., “pre-FS”) inflammatory events. We were able to examine disease activity in a number of FS and RA patients (Table 5), and were able to confirm this finding. This reduction in synovitis after the transition to FS suggests that a change in cytokine pattern may have occurred. This is indirect support for the TH1/ TH2 hypothesis discussed above. It would bring W F S into the same immunopathologic framework as that proposed for lepromatous and tuberculoid leprosy (17). One previous study found correlations between RA duration and age with the percentage expression of CD3+,CD57+ (18). The suggestion was that chronic inflammation led to the increased frequency of these cells. In that study, the raw data were logarithmically transformed before performing linear regression analysis. There was also a mean age difference of 19 years between the normal subjects and the RA patients. We thought that it was more appropriate to use Spearman’s rank correlation coefficient to analyze this question. Table 4 shows that no such correlation was found in any of the patient groups, despite the fact that the patients’ mean age and mean CD57/CD3 percentage expression were similar in the 2 studies. Furthermore, in order to make a direct comparison with the study of Dupuy d’Angeac et a1 (18), we also examined our data using regression analysis following logarithmic transformation. There was again no significant association for CD57/CD3% expression and either age (P = 0.73) or the duration of RA (P = 0.44) in the 23 RA patients. One explanation for this finding would be if disease activity were closely related to CD57+ expression on CD3+ cells, leading to a loss of the direct relationship with age in this sample. Although we were unable to test this directly from a clinical assessment, none of the laboratory markers of active disease (elevated ESR, neutrophil count, and platelet count) showed a significant correlation with CD3+ ,CD57+ percentage expression. The LGL patient group has this potential relationship with RA duration and age uncoupled by the LGL expansions, while in the FS group, the lymphopenia may be responsible, although a correlation with age was maintained. Clearly, disease dura- HEMATOLOGY OF FS AND LGL SYNDROME tion and patient age are related variables, and it is important to ensure that an increase with disease duration is not due to the patient’s increasing age. In conclusion therefore, despite the close association between FS and the LGL syndrome, evidence of a gradation from FS to the LGL syndrome was not seen in this study, thus favoring the hypothesis that a “transforming event” is required. Both those with FS alone and those with FS with the LGL syndrome had low CD4 counts, and while this may contribute to the increase in infections seen in FS, it may imply further similarities in the pathogenic mechanisms between these 2 variants of FS. The findings of this study do not exclude the hypothesis that all FS patients, including those without evidence of LGL expansions, share a common process, but they do suggest a discrete event that discriminates these 2 subgroups. Recent studies in a mouse model of autoimmunity, the MRL-lpr mouse (19), have determined that an abnormality in a single gene coding for the Fas protein leads to an autoimmune syndrome with similarities to systemic lupus erythematosus and RA. 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