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A controlled study of lymphocyte subsets in rheumatoid arthritis
CLINICAL
IMMUNOmY
AND
IMMUNOPATHOLOGY
Vol. 63, No. 3, June, pp. 237-244,
A Controlled
1992
Study of Lymphocyte
Subsets in Rheumatoid
Arthritis
CHARLESW. CALDWELL *,‘r2 ALAN J. BRIDGES,?SARA E. WALKER,?+ KAREN L. SNIARR,§R. JACK REICHERT,~ SHARONK. ANDERSON;’JOHN E. HEWETT;’ AND JERRY C. PARKER§ Departments
of
*Pathology and. fMedicine, and the ‘Biostatistics Group, University of Missouri School of Medicine and $Medicine Service and $Psychology Service, Harry S. Truman Memorial Veterans Hospital, Columbia, Missouri 65203
The purpose of this study was to determine if patients with rheumatoid arthritis WA) exhibited unique patterns of peripheral blood lymphocyte (PBL) subsetsin comparison to patients with osteoarthritis (OA) and, further, if such differences related to disease activity or nondiseasefactors. Data from 63 RA patients and 47 OA patients revealed that the RA patients had lower absolute numbers of CD2’ and CD4+ lymphocytes. Small differences also were found in selected B-cell subsetsand subsetsof lymphocytes expressing CD16 and/or CD57 antigens. Further analysis revealed that these differences were due primarily to the effects of cytotoxic medications in the RA group. However, there were also alterations in some subsets independent of medication groups. PBL subsetsin RA patients did not relate to chronic low-dose prednisone or measures of disease activity. This study demonstrated the need to control carefully for variables such as age and medication in immunophenotypie investigations of RA. 6 1~ Academic PRESS, I~C. INTRODUCTION
Rheumatoid arthritis (RA) is a systemic illness of unknown etiology associated with a wide array of immunologic abnormalities (1, 2). Numerous studies have examined the immunophenotypes of peripheral blood mononuclear (PBM) cells from patients with RA (3-18). Assessment of lymphocytes from an accessible compartment such as the blood may reflect disease activity, but studies designed to produce support for this supposition have yielded inconsistent results (3,4,6,7, 9-11, 13, 16, 17). Such inconsistency might be accounted for by differences in disease assessment, sample sizes, control population, and various medications. It has been proposed that medications which modulate 1 To whom correspondence should be addressed at Department of Pathology, Ellis Fischel Cancer Center, University of Missouri Health Science Centers, 115 Business Loop 7OW, Columbia, MO 65203. 2 Grant support: Medical Research Service of the Veterans Administration and by a grant from the National Institute of Disability and Rehabilitation Research (H133B80075).
the immune system alter lymphocyte subsets in patients with RA, but evidence to support this hypothesis is limited (3, 10-14). The purpose of this study was to compare peripheral blood lymphocytes (PBL) of RA patients with those of a demographically similar group of osteoarthritis (OA) patients without autoimmune disease to determine if RA patients demonstrated specific alterations in immune cellular subsets and if such alterations were related to disease activity or to other immune modifiers. MATERIALS
AND METHODS
Subjects
One hundred and twenty-seven male, Caucasian patients with RA or OA were studied prospectively at the Harry S. Truman Memorial Veterans Hospital. Although these diseases also occur in women and nonCaucasian males, it was felt that limiting the study group would control for additional levels of heterogeneity. Informed consent was obtained in compliance with the Institutional Review Board of the University of Missouri-Columbia. Eighty-one percent of eligible patients who were asked to participate in the study gave their consent. The RA patients (n = 80) were diagnosed with classic or definite RA on the basis of American Rheumatism Association (ARA) criteria (19). These patients were followed regularly in a rheumatology clinic. The RA patients were taking one, or a combination of, nonsteroidal anti-inflammatory drugs (NSAID), and/or prednisone, and/or a disease-modifying anti-rheumatic drug (DMARD). For statistical analyses, the DMARDs were grouped into the noncytotoxic category (i.e., gold, penicillamine, and hydroxychloroquine) or the cytotoxic category (i.e., methotrexate, cyclophosphamide, and azathioprine). RA patients were removed from the study if their medication category changed from noncytotoxic to cytotoxic during the course of the investigation (n = 17). Ninety-four percent of the remaining RA patients (n = 63) had been treated with a stable
237
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238
CALDWELL
medication regimen for at least 6 months before entering the study. The mean age of the RA sample (n = 63) was 60.7 years (SD = 6.6), and the mean time from initial diagnosis was 13.3 years. The distribution of RA patients by Steinbrocker functional class (20) was: Class I, n = 8; Class II, n = 46; and Class III, n = 9. The control group consisted of 47 OA patients recruited from the VAH Orthopedic Clinic and selected because they did not have an autoimmune disease. The diagnostic criteria for OA were (1) clinical involvement of one or more joints and (2) radiographic evidence of at least two of the following: (a) loss of articular cartilage, (b) reactive changes at the margins of joints and subchondral bone, or (c) osteophyte formation. OA patients were excluded from the study if the involved joint had been replaced. The mean age of the OA sample was 64.5 years (SD = 6.7), and the mean time from initial diagnosis was 15.9 years. All OA patients received treatment with NSAIDs throughout the study. Clinical Measures
Measurements of RA disease status, including total active joint counts (21) and estimates of morning stiffness, were performed by the same experienced rheumatology nurse clinician throughout the study. Measurements of disease status were obtained on three representative occasions (i.e., baseline, 3 months, and 6 months). To obtain representative values for each individual subject over a 6-month time frame the three data points were averaged. A mean was calculated for total active joint count and morning stiffness for data analysis purposes. Immunophenotypic
Analysis
Peripheral blood was obtained on three separate occasions (i.e., baseline, 3 months, and 6 months) from
-.- .~~
ET AL.
RA and OA patients by standard venipuncture methods. The three results from each patient were averaged to “smooth out” minor fluctuations and to obtain a representative value for each subject over a g-month time frame. (The CD4/CD8 ratio was calculated at each data point. The three ratios were averaged to yield a mean CD4/CD8 ratio per subject.) There were no missing data points. The specimens were obtained between 9:00 and 11:00 AM to control for circadian rhythm (22). PBL were isolated by density-gradient centrifugation (23, 24). We have previously reported no significant differences in percentages of CD2’ (P = 0.40) and CD4’ (P = 0.38) lymphocytes in a comparison of density-gradient separation and whole blood lysis, with a marginal difference in CD8’ cells (P = 0.04) (23). The fluorochrome-conjugated fluorescein isothiocyanate (FITC) or phycoerythrin (PE) monoclonal antibodies (MoAb) used in this study, with their major reactivities, are listed in Table 1 (25). The technical aspects of immunostaining have been reported previously (23. 24). PBL subsets values were obtained by analysis of dual-parameter histograms, each containing 10,000 cells. Isotypic negative control MoAbs, labeled with FITC, or PE, were used to determine the negativei positive cutoff point. Percentages of cells reactive (or nonreactive) with each pair of MoAbs were determined using the QUADSTAT program (Coulter Electronics). Percentages of MoAb positivity within the “lymphocyte” light scatter gate for each MoAb were adjusted based on the CD45 “bright” and CD14 positivity in order to compensate for erythrocytes, neutrophils, and monocyte contamination of this light scatter region (23). These corrected percentages were then used to determine the absolute number of MoAb-positive cell subsets by multiplying the total lymphocyte count obtained from a standard hematology profile by each corrected subset percentage.
TABLE 1 Monoclonal Antibodies
CD”
MoAbb
Main reactivity; other reactivity
Source’
CD2 CD4 CD5 CD8 CD14 CD16 CD20 CD21 CD45 CD57 NC.’
CCT 11 CC T4 Leu 1 cc T8 Leu M3 Leu 11 Leu 16 B2 HLe-1 Leu 7 HLA DR
T cells (SRBC receptor); NK cells Helper/inducer T cells T cells (strong); subset of B cells (weak) Suppressor/cytotoxic T cells; NK cells Monocytes; granulocytes Natural killer cells, granulocytes, monocytes B-cell restricted Mature B cells All leukocytes Natural killer cells; T-cell subset Class II major hi&compatibility antigen; B cells, monocytes, “activated” T cells and NK cells
c C CD C BD BD BD C BD BD BD
Fluorochrome”
__a Clusters of differentiation (CD) as defined by the Fourth International Workshop on Leukocyte Antigens, Vienna, 1989 (25). ’ Monoclonal antibody. c C, Coulter Immunology, Hialeah, FL; BD, Becton-Dickinson, Mountain View, CA. d P, phycoerythrin; F, Fluorescein isothiocyanate. e Not clustered.
P P P F P P F P F F F
LYMPHOCYTE
Data Analysis The first step in data analysis was to examine the normality of the population distribution of each immunophenotypic measure. For most measures, the normality assumption was not met, so nonparametric procedures based on ranks (if readily available) were used in subsequent comparisons. Second, analysis of covariante (ANCOVA) was used to adjust the data for agerelated group differences which occurred on a few selected measures. Third, immunophenotypic values from the RA and OA groups were compared using the Wilcoxon rank sum test. Fourth, disease activity measures were correlated with the immunophenotypic subsets using Spear-man correlation coefficients to determine if disease activity was related to any observed group differences. Fifth, the PBL subset values were compared for RA patients taking cytotoxic medications, RA patients taking noncytotoxic medications, and the OA patients (Kruskal-Wallis test). Sixth, the effect of prednisone on immunophenotypic subsets was analyzed using the Wilcoxon rank sum test. Due to the large number of statistical comparisons, the significance level was adjusted to 0.01. RESULTS
Age Comparisons
for RA and OA Patients
Since age differences the OA subjects (P = tion coefficients were tionships between age
239
SUBSETS IN RA
were found between the RA and 0.007), Spearman rank correlaused to examine possible relaand each of the immunophenoTABLE
typic measures. Age was found to be correlated with the total lymphocyte count and with absolute numbers of CD2’ and CD4+ lymphocytes. Age also correlated with relative percentages of lymphocytes positive for CD2+ and CD8+, as well as CD57+/CD16-, CD57-/ CD16’, and CD57+/CD16+ NK cells. Therefore, subsequent comparisons of these specific immunophenotypic measures were accomplished with ANCOVA using age as a covariate. Lymphocyte
Subsets of RA and OA Patients
The relative and absolute values for lymphocyte subsets of RA and OA patients are shown in Table 2. Statistically significant differences in percentages of cellular subsets between the two groups were found for CD5+/CD20+ B cells (P = 0.0002), CD20+/CD21B cells (P = O.OOOl>, CD57+/CD16lymphocytes (P = 0.003), and CD57+/CD16+ lymphocytes (P = 0.007). In all of these subsets, the magnitude of the observed difference was small, so the biological importance of each statistically significant difference is uncertain. The total lymphocyte count differed significantly between the groups (P = 0.0006; RA = 1309 cell+l; OA = 1598 cells/pi). Significant differences were found between patient groups in absolute numbers of CD2+ lymphocytes (P = 0.002), CD4+ lymphocytes (P = 0.0005), CD20+ B cells (P = 0.007), and CD20f/ CD21- B cells (P = 0.0001). Overall, the most notable numerical differences between the groups were observed in the decreased absolute numbers of CD4+ lymphocytes in the RA group, which in turn was re2
Comparison between RA and OA Patients
on Lymphocyte
RAb Immunophenotypic
subsets
Total lymphocyte count CD2 + lymphocytes’ CD4 + helper/inducer cells CD8 + suppressorkytotoxic cells CD4/CD8 ratid “Activated” CD2 + lymphocytes (CD2 +/HLA-DR+ Class II MHC antigen (HLA-DR) Total B cells (CD20) CD5 + /CD20 + B cells CD20+/C!D21- B cells CD20+/CD21+ B cells CD57+/CD16lymphocytes CD57-/CD16+ lymphocytes CD57+/CD16+ lymphocytes ; gr6y
of initial,
)
Subsets” OA’
P valued
Absolute number
Percent
Absolute number
Percent
Absolute number
1309 1062 670 283 3.6 50 185 96 21 23 74 351 139 82
80.4 51.6 21.4 3.6 4.1 14.5 7.4 1.6 1.8 5.6 26.3 11.6 6.6
1598 1284 829 296 3.4 46 206 132 17 57 75 327 161 75
80.4 52.0 18.6 3.4 3.1 13.2 8.1 1.0 3.5 4.6 20.6 10.5 4.8
0.0006* 0.002* 0.0005* 0.37 0.39 0.84 0.08 0.007 0.04 0.0001 0.79 0.78 0.13 0.89
Percent 0.99* 0.91 0.04* 0.39 0.05 0.36 0.19 0.0002 0.0001 0.12 0.003* 0.11* 0.007*
3-month, and 6-month measures for each subject expressed as a group mean.
‘N = 47: d Data not normally distributed, therefore Wilcoxon rank sum nonparametric e N = 46 for OA group due to unusable lab data for one patient. ‘Ratio of percentages of CD4 and CDS. * ANCOVA age-adjusted P values for group differences.
P values are reported, except for age-adjusted comparisons.
240
CALDWELL
fleeted in the decreased total number cytes. Correlation of Lymphocyte Disease Activity
of CD2 + lympho-
Subsets, and RA
Within the RA sample, Spear-man correlations were used to examine relationships between measures of RA disease activity and the absolute values of the PBL subsets where significant differences in absolute values were found between RA and OA patients (Table 3). Interestingly, there were no significant correlations between the disease activity measures (i.e., total active joint count, morning stiffness) and the PBL subsets for which group differences were found (i.e., total lymphocyte count, CD2+ lymphocytes, CD4+ lymphocytes, CD20+/CD21B cells). Thus, the observed PBL subset differences between the RA subjects and the OA subjects did not appear to be accounted for by variations in disease activity. Modulation of Lymphocyte Cytotoxic Therapy
Subsets by
To explore other possible reasons for differences in PBL subsets between the RA and the OA subjects the relationship between lymphocyte subsets and treatment with DMARDs was analyzed (Table 4). The hypothesis was that treatment of RA patients with cytotoxic agents might account for the lower PBL counts. Thus, the 63 RA patients taking DMARDs were divided into two groups. Medication group 1 (MGl) was composed of 42 RA patients receiving noncytotoxic treatment with gold (n = 271, penicillamine (n = lo), or hydroxychloroquine (n = 4). One additional MGl patient was taking hydroxychloroquine at baseline, hydroxychloroquine at the 3-month assessment, and gold at the 6-month assessment. Medication group 2 (MG2) was composed of 21 patients receiving cytotoxic treatment with methotrexate (n = 91, azathioprine (n = 91, or cyclophosphamide (n = 3). Two of the MG2 TABLE3 Correlation between Measures of RA Disease Activity and Absolute Numbers of Lymphocyte Subsets”
Total lymphocyte count TAJCb MS’
CD2 + lymphocytes
V3.M
- 0.04 -0.11
Note. All P values Averages of initial, measures. a Cells per microliter. b Total active joint ’ Morning stiffness
- 0.08 - 0.09
CD4+ helper/ inducer cells (CD4) 0.04 -0.12
patients were taking cyclophosphamide at baseline, methotrexate at the 3-month assessment, and cyclophosphamide at the g-month assessment. One MG2 patient was taking methotrexate at baseline, methotrexate at the 3-month assessment, and azathioprine at the 6-month assessment. The relative and absolute values for the lymphocyte subsets in MGl, MG2, and the OA group are shown in Table 4. Differences in the absolute counts between the RA and the OA groups were due primarily to the lower absolute lymphocyte counts in the subgroup of RA patients receiving cytotoxic medications (MG2). Specifically, MG2 differed significantly from the OA patients in absolute numbers of CD2’ lymphocytes, CD4+ lymphocytes, HLA-DR+ lymphocytes, CD20’ B cells, CD20+/CD21B cells, and CD20C/CD21 c B cells; whereas MGl differed from the OA patients only on absolute numbers of CD4’ lymphocytes, CD2O.Y CD21 - B cells, and CD20+/CD21+ B cells. Regarding percentages of PBLs, both MGl and MG2 differed significantly from the OA patients in CD5+/CD20t B cells, CD20+/CD21B cells, and CD57”/CD16lym. phocytes. Differences in disease activity between MGI and MG2 did not appear to account for the observed differences in PBL subsets because Wilcoxon rank sum tests revealed that MGl and MG2 did not differ in total active joint counts or estimates of morning stiffness. Effects of Prednisone
on Lymphocyte
Subsets
To more fully delineate the impact of medication on PBL subsets, the RA patients receiving low-dose prednisone (~10 mg/dayJ were analyzed separately within both MGl and MG2 (data not shown). There were 11 patients receiving prednisone in MGl and 9 patients receiving prednisone in MG2. The data revealed that there were no significant differences in lymphocyte subsets between those RA patients receiving low-dose prednisone and those not receiving prednisone. This observation suggested that chronic low-dose prednisone did not influence the lymphocyte subsets in the peripheral blood of patients with RA. It should be pointed out, however, that the statistical power of this analysis should be considered given the low number in each group. DISCUSSION
CD20+/CD21-B cells 0.19 - 0.20
for Spearman correlations are >O.lO. 3-month, and 6-month values were used for all
count. (hours).
ET AL.
Because RA is an autoimmune disease, it is rational to consider the possibility that alterations of immune cellular subsets might be valuable from an accessible compartment such as the blood and possibly provide useful information related to immune status or disease activity. Clearly, most of the pathology, and probably the traffiking of lymphoid cells, is aimed at the inflamed synovium. However, if lymphoid cells in peripheral blood are a direct (or indirect) reflection of disease activity, then evaluation of this easily accessible com-
LYMPHOCYTE
Comparisons among RA Medication
TABLE 4 Subgroups and OA Group on Lymphocyte Subsets”
RA MGlb Immunophenotypic
subsets
Total lymphocyte count CD2+ lymphocytes Helper/inducer cells (CD41 Suppressor/cytotoxic cells (CDS) CD4/CD8 ratio’ “Activated” CD2+ lymphocytes (CD2+/HLA-DR+) Class II MHC antigen (HLA-DR) Total B cells (CD201 (from CD20f/CD21+) CD5 + /CD20+ B cells CD20’/CD21B cells CD20+/CD21+ B cells CD57+/CD16lymphocytes CD57-/CD16+ lymphocytes CD57+/CD1GC lymphocytes
241
SUBSETS IN RA
RA MG2’
Absolute number
%
1448 1160 746 293 3.6 55 214 115 23 27 87 373 151 82
80.0 51.6 20.5 3.6 4.0 14.8 7.8 1.6 1.8 6.0 26.2 11.4 6.2
Absolute number 1031 866 524 262 3.8 40 125 59 17 13 46 307 117 81
OAd % 81.2 51.8 23.2 3.8 4.2 13.9 6.5 1.6 1.7 4.8 26.7 12.0 7.2
Absolute number 1598 1284 829 296 3.4 46 206 132 18 57 75 327 161 75
P
% 8;4 52.0 18.6 3.4 3.1 13.2 8.1 1.0 3.5 4.6 20.6 10.5 4.8
valuee
Absolute number
%
0.0001*~~ 0.0005*f6
0.86*
O.OOO1*f~~h
0.99
0.30 0.66 0.04
0.26 0.66 0.13 0.32
0.0004f~ 0.0002fg
0.09
0.05 0.0001~~~ O.OOwh 0.14 0.02 0.41
0.0007g*h 0.0001~~~ 0.05 O.OlM,h 0.27* 0.02*
a Averages of initial, 3-month, and 6-month measures for each subject expressed as a group mean. b RA patients taking gold, penicillamine, or hydroxchloroquine (N = 42). ’ RA patients taking methotrexate, azathioprine, or cyclophosphamide (N = 21). d OA patients taking nonsteroidal anti-inflammatory drugs (N = 47). e Data not normally distributed, therefore nonparametric Kruskal-Wallis P values are reported, except for age-adjusted comparisons. The P values represent comparisons of all three groups. f Post-hoc comparisons revealed RA MGl significantly different from RA MG2. 8 Post-hoc comparisons revealed RA MG2 significantly different from OA group. h Post-hoc comparisons revealed RA MGl significantly different from OA group. ’ Ratios of percentages of CD4 and CD8 were calculated for each subject and then averaged over the three time points to yield a mean ratio per subject. These numbers were then averaged over the entire group to yield the mean CD4XD8 ratio reported above. *ANCOVA age-adjusted P values for group differences.
partment might be useful. It is not clear that alterations in lymphocyte subsets reported in earlier studies reflect disease activity or if they are secondary to medications or other intervening factors. In this study, we examined peripheral blood lymphocyte subsets from a large group of patients with RA or OA. Patients with RA had lower absolute numbers of total lymphocytes, total CD2+ lymphocytes, CD4+ lymphocytes, total CD20f B cells, and CDZO+/CDZlB cells (all P < 0.002). Differences in total lymphocytes and total CD2+ lymphocytes were associated with cytotoxic drug therapy used by some of the RA patients (Table 4). Interestingly, there were no significant correlations between PBL subsets and either RA disease activity or the use of low-dose prednisone within the RA groups. These results suggested that chronic therapy with DMARDs in RA may be associated with PBL subset abnormalities independent of RA disease activity. Statistically significant differences were also found between the RA and OA groups in some of the quantitatively smaller lymphocyte subsets. Several investigators have reported significant differences between cellular subsets of patients with active or inactive RA (3,4,6,7,9, lo), but the differences between “active” and “inactive” categories were minimal (or not reported) in some studies and significant interstudy variability in classification was present.
These incongruities, coupled with large within-subject variability in lymphocyte subset values, make clinically useful interpretations of the data difficult. Additionally, medications are known to alter lymphocyte subsets and thus may have obscured real differences in the distribution of lymphocyte subsets related to disease activity. To overcome these problems, comprehensive assessments of disease activity were performed, and statistical analyses were used to evaluate known or suspected intervening factors, including medications. In this study, there were no statistically significant correlations between disease activity measures and the PBL subset values on which group differences were found. However, differences in subset values between RA and OA patients were obvious and thus required further analysis of medication effects. Many patients with RA receive cytotoxic medications which have been shown to produce lymphocytopenia (26-30). Weekly pulse-dose methotrexate therapy in patients with RA has been shown to produce a modest decrease in the total lymphocyte count after only 13 weeks of therapy, but with relative increases in the percentage of total T cells and CD4+ lymphocytes (26). In another short-term study on the efficacy of methotrexate in RA, no significant differences in the PBL subsets were found in 12 weeks (27). However, a longterm study (2 years) revealed increased percentages of
242
CALDWELL
total T cells (CD3) and T-helper cells (CD4) compared to baseline values (281, which is consistent with the 13-week study (26). Cytotoxic therapy with azathioprine and cyclophosphamide also has been associated with lower total lymphocyte counts, but not to changes in the relative percentages or absolute numbers of T or B cells (29, 30). Our study, in which 94% of the RA patients had been treated with stable doses of cytotoxic therapy for at least 6 months, produced statistically significant differences in the total lymphocyte count. Second, significant differences were observed in absolute numbers of the CD2+ T cells, CD4+ T-helper cells, CD20+ B cells, and HLA-DR+ lymphocytes, but not in percentages of lymphocytes when the cytotoxic and noncytotoxic groups of RA patients were compared. This is consistent with earlier studies in which a “control” group of RA patients treated with NSAIDs, prednisone, or gold was investigated (29, 30). These patients had significantly higher total lymphocyte counts than patients taking cytotoxic agents, but were still below that of normal controls. These findings are in accord with the current study of RA patients in each medication group and OA controls. Beyond these medication-related findings, both groups of RA patients differed from the OA controls in other subsets. Corticosteroid therapy, which is also known to induce lymphocytopenia (31331, is commonly used in conjunction with other DMARDs. Therefore, sustained modulation of cellular subsets by the chronic use of these agents in diseases of abnormal immune regulation is of interest. In low oral doses, corticosteroids appear to produce only transient lymphocytopenia in normal subjects, with CD4+ T-helper cells an CD2+ T cells affected to a much greater extent that CD8+ T-suppressor cells (31). Similar transient responses to prednisone (lo-mg dose) have been found in a small group of patients with RA, although CD3, rather than CD2, was used to measure T cells (32). The use of lowdose prednisone in our study was found not to contribute to subset changes, but this finding may be due to the fact that prednisone was used long term in the present study as compared to short-term usage in other studies (31-33). Male OA patients receiving stable doses of NSAIDs were selected as the control group for this study. Comparison of the PBL subsets from the control patients in this study with the data from control subjects from other studies revealed comparable values (4-6). Thus, the findings from this study did not appear to be attributable to characteristics of the OA control sample. The four cellular subsets (CD5+/CD20+ B cells, CD20+/CD21B cells, CD57+/CD16’ NK cells, and CD57+/CD16lymphocytes) that were found to differ in percentage from OA controls in both the cytotoxic and noncytotoxic groups of RA are of interest. Since the same alterations occurred in both groups of RA pa-
ET AL.
tients, consideration must be given to the possibility that these changes are disease related. For instance, CD5 +/CD20+ B cells are known to be increased in RA and have been shown to be committed to production of IgM with rheumatoid factor activity (34, 35). The percentages of CD5 + B cells in the present study are consistent with those previously reported (35). However, in the present study, the percentage of CD20C/CD21 B cells was decreased in the RA patients. This contrasts with studies which show a reduction of CD21 from normal B cells following in vitro B-cell activation and an increase in CD20+/CD21B cells in diseases characterized by B-cell activation and hypergammaglobulinemia (36, 37). Since B cells account for only 78% of the total lymphocytes, the significance of these findings is uncertain. It should also be noted that somewhat different numbers of total B cells were measured when using the CD20-containing MoAb pair Bl/B2 compared to Leu lG/Leu 1. However, the percentage of CD20’ cells should be the same with either MoAb. The reason(s) for such differences is not entirely clear. Data from this laboratory demonstrate that while these two MoAb bind to the same antigen on human B cells, cells from squirrel monkeys bind only Bl, suggesting a difference in epitopic-binding sites (unpublished data). The differences found in the present study were small, ranging from 0.6 to 2.2%, were not statistically significant (P > 0.051, and were found to correlate well (Pearson coefficient, 0.88 for absolute CD20). Thus, the minor discrepancy in number of total B cells measured may be related to reagents used, experimental conditions, or possibly epitopic differences in some individuals. The differences in the lymphocyte subsets defined by CD57 and CD16 reactivity are also not well explained (38). Since CD57 is present on a subgroup of CD2+ T cells as well as NK cells, the CD57+/CD16subgroup may include this subset of T cells accounting for some of the differences. It should be understood that RA is a heterogeneous disorder with variable immunologic dysfunction, suggesting that differences in smaller subsets may fall within the spectrum of immune abnormalities in RA. In conclusion, this study revealed that most, but not all, of the changes in PBL subset values found in RA patients were probably the result of therapy utilizing DMARDs. There were no significant correlations between PBL subsets and RA disease activity, thus suggesting that chronic therapy with DMARDs in RA may be associated with PBL subset abnormalities independent of the disease activity of RA, but still related to the presence of the disease. A variety of differences were found among the smaller lymphocyte subsets which may be disease related; however, the clinical significance of these findings is unclear. Conflicting results from previous studies may have resulted from methodological differences or failure to account for rel-
LYMPHOCYTE
evant patient characteristics logical effects.
or important
SUBSETS
pharmaco-
ACKNOWLEDGMENTS
The authors gratefully acknowledge the contributions of Barb Cullen, Thomas Feldbush, Kristofer J. Hagglund, Robert Hoffman, Frank O’Sullivan, Gordon Sharp, and Elaine Smith.
15. 16.
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IN RA
243
teristics of peripheral blood and synovial fluid lymphocytes from patients with rheumatoid arthritis. Stand. J. Zmmurwl. 7,405417,1978. Nilsson, E., and Biberfeld, G., T lymphocyte subpopulations defined by monoclonal antibodies in synovial fluid of patienta with rheumatic disease. Clin. Lab. Zmmunol. 9, 93-97, 1982. Duclos, M., Zeidler, H., Liman, W., Pichler, W. J., Rieber, P., and Peter, H. H., Characterisation of blood and synovial fluid lymphocytes from patients with rheumatoid arthritis and other joint diseases by monoclonal antibodies (OKT Series) and acid a-naphthyl e&erase staining. Rheumatol. Znt. 2, 75-82, 1982. Bertouch, J. V., Roberts-Thomson, P. J., Brooks, P. M., and Bradley, J., Lymphocyte subsets and inflammatory indices in synovial fluid and blood of patients with rheumatoid arthritis. J. Rheumntol. 11, 754-759, 1984. Pitzalis, C., Kingsley, G., Murphy, J., and Panayi, G., Abnormal distribution of the helper-inducer and suppressor-inducer T-lymphocyte subsets in the rheumatoid joint. Clin. Zmmunol. Zmmunopathol. 45, 252-258, 1987. Ropes, M. W., Bennett, G. A., Cobb, S., Jacox, R., and Jessar, R. A., 1958 Revision of diagnostic criteria for rheumatoid arthritis. Bull. Rheum. Dis. 9, 175-176, 1958. Steinbrocker, O., Traeger, C. H., and Batterman, R. C., Therapeutic criteria in rheumatoid arthritis. JAMA 140, 659-662, 1949. American Rheumatism Association, “Dictionary of the Rheumatic Diseases,” Vol. I, “Signs and Symptoms,” pp. l-87, Contact Associates International Ltd., New York, 1982. Ritchie, A. W., Oswald, I., Micklem, H. S., Boyd, J. E., Elton, R. A., Jazwinska, E., and James, K., Circadian variation of lymphocyte subpopulations: A study with monoclonal antibodies. Br. Med. J. 266, 17731775, 1983. Caldwell, C. W., Maggi, J., Henry, L. B., and Taylor, H. M., Fluorescence intensity as a quality control parameter in clinical flow cytometry. Am. J. Clin. Pathol. 88, 447456, 1987. Caldwell, C. W., and Patterson, W. P., Relationship between T200 antigen expression and stages of B cell differentiation in resurgent hyperplasia of bone marrow. Blood 70, 1165-1172, 1987. Knapp, W., D&ken, B., Rieber, P., Schmidt, R. E., Stein, H., and von dem Brone, A. E. G. Kr., CD antigens 1989. Blood 74,14481450,1989. Andersen, P. A., West, S. G., O’Dell, J. R., Via, C. S., Claypool, R. G., and Kotzin, B. L., Weekly pulse methotrexate in rheumatoid arthritis. Ann. Intern. Med. 103, 489496, 1985. Weinblatt, M. E., Coblyn, J. S., Fox, D. A., Fraser, P. A., Holdsworth, D. E., Glass, D. N., and Trentham, D. E., Efficacy of low-dose methotrexate in rheumatoid arthritis. N. Engl. J. Med. 312, 818-822, 1985. Weinblatt, M. E., Trentham, D. E., Fraser, P. A., Holdsworth, D. E., Falchuk, K. R., Weissman, B. N., and Coblyn, J. S., Longterm prospective trial of low-dose methotrexate in rheumatoid arthritis. Arthritis Rheum. 31, 167-175, 1988. Yu, D. T., Clement-s, P. J., Peter, J. B., Levy, J., Paulus, H. E., and Barnett, E. V., Lymphocyte characteristics in rheumatic patients and the effect of azathioprine therapy. Arthritis Rheum. 17, 3745, 1974. Clements, P. J., Yu, D. T. Y., Levy, J., Paulus, H. E., and Barnett, E. V., Effects of cyclophosphamide on B- and T-lymphocytes in rheumatoid arthritis. Arthritis Rheum. 17, 347-353, 1974. Slade, J. D., and Hepburn, B., Prednisone-induced alterations of circulating human lymphocyte subsets. J. Lab. Clin. Med. 101, 479-487,1983. Hermanns, P., Verbruggen, G., Veys, E. M., Vansteenkiste, M.,
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Delanghe, J., and Mielants, H., Influence of low dose corticosteroid administration on the lymphocytes subpopulations. J. Rheumatol. 9, 648-649, 1982. 33. Zweiman, B., Atkins, P. C., Bedard, P. M., Flaschen, S. L., and Lisak, R. P., Corticosteroid effects on circulating lymphocyte subset levels in normal humans. J. Clin. Zmmunol. 4, 151-155, 1984. 34. Casali, P., Burastero, S. E., Nakamura, M., Inghirami, G., and Notkins, A. L., Human lymphocytes making rheumatoid factor and antibody to ssDNA belong to Leu-1 + B-cell subset. Science 236, 77-80, 1987. 35. Hardy, R. R., Hayakawa, K., Shim&u, M., Yamasaki, K., and Kishimoto, T., Rheumatoid factor secretion from human Leu-1 + B cells. Science, 236, 81-83, 1987. Received October 23, 1991; accepted with revision February 28, 1992
ET AL 36. Anderson, K. C., Roach, J. A., Daley, J. F., Schlossman, S. F., and Nadler, L. M., Dual fluorochrome analysis of human B lymphocytes: Phenotypic examination of resting, antiimmunoglobulin stimulated, and in vivo activated B cells. J Zmmunol. 136, 3612-3618, 1986. 37. Boyd, A. W., Anderson, K. C., Freedman, A. S., Fisher, D. C., Slaughenhoupt, B., Sehlossman, S. S., and Nadler, L. M., Studies of in vitro activation and differentiation of human B lymphocytes. I. Phenotypic and functional characterization of the B cell population responding to anti-Ig antibodies. J. Zmmunol. 134. 15161523, 1985. 38. Nagler, A., Lanier, L. L., Cwirla, S., and Phillipe, J. H., Comparative studies of human FcRIII-positive and negative natural killer cells. J. Zmmunol. 143, 3183-3191, 1989.
IMMUNOmY
AND
IMMUNOPATHOLOGY
Vol. 63, No. 3, June, pp. 237-244,
A Controlled
1992
Study of Lymphocyte
Subsets in Rheumatoid
Arthritis
CHARLESW. CALDWELL *,‘r2 ALAN J. BRIDGES,?SARA E. WALKER,?+ KAREN L. SNIARR,§R. JACK REICHERT,~ SHARONK. ANDERSON;’JOHN E. HEWETT;’ AND JERRY C. PARKER§ Departments
of
*Pathology and. fMedicine, and the ‘Biostatistics Group, University of Missouri School of Medicine and $Medicine Service and $Psychology Service, Harry S. Truman Memorial Veterans Hospital, Columbia, Missouri 65203
The purpose of this study was to determine if patients with rheumatoid arthritis WA) exhibited unique patterns of peripheral blood lymphocyte (PBL) subsetsin comparison to patients with osteoarthritis (OA) and, further, if such differences related to disease activity or nondiseasefactors. Data from 63 RA patients and 47 OA patients revealed that the RA patients had lower absolute numbers of CD2’ and CD4+ lymphocytes. Small differences also were found in selected B-cell subsetsand subsetsof lymphocytes expressing CD16 and/or CD57 antigens. Further analysis revealed that these differences were due primarily to the effects of cytotoxic medications in the RA group. However, there were also alterations in some subsets independent of medication groups. PBL subsetsin RA patients did not relate to chronic low-dose prednisone or measures of disease activity. This study demonstrated the need to control carefully for variables such as age and medication in immunophenotypie investigations of RA. 6 1~ Academic PRESS, I~C. INTRODUCTION
Rheumatoid arthritis (RA) is a systemic illness of unknown etiology associated with a wide array of immunologic abnormalities (1, 2). Numerous studies have examined the immunophenotypes of peripheral blood mononuclear (PBM) cells from patients with RA (3-18). Assessment of lymphocytes from an accessible compartment such as the blood may reflect disease activity, but studies designed to produce support for this supposition have yielded inconsistent results (3,4,6,7, 9-11, 13, 16, 17). Such inconsistency might be accounted for by differences in disease assessment, sample sizes, control population, and various medications. It has been proposed that medications which modulate 1 To whom correspondence should be addressed at Department of Pathology, Ellis Fischel Cancer Center, University of Missouri Health Science Centers, 115 Business Loop 7OW, Columbia, MO 65203. 2 Grant support: Medical Research Service of the Veterans Administration and by a grant from the National Institute of Disability and Rehabilitation Research (H133B80075).
the immune system alter lymphocyte subsets in patients with RA, but evidence to support this hypothesis is limited (3, 10-14). The purpose of this study was to compare peripheral blood lymphocytes (PBL) of RA patients with those of a demographically similar group of osteoarthritis (OA) patients without autoimmune disease to determine if RA patients demonstrated specific alterations in immune cellular subsets and if such alterations were related to disease activity or to other immune modifiers. MATERIALS
AND METHODS
Subjects
One hundred and twenty-seven male, Caucasian patients with RA or OA were studied prospectively at the Harry S. Truman Memorial Veterans Hospital. Although these diseases also occur in women and nonCaucasian males, it was felt that limiting the study group would control for additional levels of heterogeneity. Informed consent was obtained in compliance with the Institutional Review Board of the University of Missouri-Columbia. Eighty-one percent of eligible patients who were asked to participate in the study gave their consent. The RA patients (n = 80) were diagnosed with classic or definite RA on the basis of American Rheumatism Association (ARA) criteria (19). These patients were followed regularly in a rheumatology clinic. The RA patients were taking one, or a combination of, nonsteroidal anti-inflammatory drugs (NSAID), and/or prednisone, and/or a disease-modifying anti-rheumatic drug (DMARD). For statistical analyses, the DMARDs were grouped into the noncytotoxic category (i.e., gold, penicillamine, and hydroxychloroquine) or the cytotoxic category (i.e., methotrexate, cyclophosphamide, and azathioprine). RA patients were removed from the study if their medication category changed from noncytotoxic to cytotoxic during the course of the investigation (n = 17). Ninety-four percent of the remaining RA patients (n = 63) had been treated with a stable
237
All
Copyright 0 1992 rights of reproduction
0090-1229/92 $4.00 by Academic F’ress, Inc. in any form reserved.
238
CALDWELL
medication regimen for at least 6 months before entering the study. The mean age of the RA sample (n = 63) was 60.7 years (SD = 6.6), and the mean time from initial diagnosis was 13.3 years. The distribution of RA patients by Steinbrocker functional class (20) was: Class I, n = 8; Class II, n = 46; and Class III, n = 9. The control group consisted of 47 OA patients recruited from the VAH Orthopedic Clinic and selected because they did not have an autoimmune disease. The diagnostic criteria for OA were (1) clinical involvement of one or more joints and (2) radiographic evidence of at least two of the following: (a) loss of articular cartilage, (b) reactive changes at the margins of joints and subchondral bone, or (c) osteophyte formation. OA patients were excluded from the study if the involved joint had been replaced. The mean age of the OA sample was 64.5 years (SD = 6.7), and the mean time from initial diagnosis was 15.9 years. All OA patients received treatment with NSAIDs throughout the study. Clinical Measures
Measurements of RA disease status, including total active joint counts (21) and estimates of morning stiffness, were performed by the same experienced rheumatology nurse clinician throughout the study. Measurements of disease status were obtained on three representative occasions (i.e., baseline, 3 months, and 6 months). To obtain representative values for each individual subject over a 6-month time frame the three data points were averaged. A mean was calculated for total active joint count and morning stiffness for data analysis purposes. Immunophenotypic
Analysis
Peripheral blood was obtained on three separate occasions (i.e., baseline, 3 months, and 6 months) from
-.- .~~
ET AL.
RA and OA patients by standard venipuncture methods. The three results from each patient were averaged to “smooth out” minor fluctuations and to obtain a representative value for each subject over a g-month time frame. (The CD4/CD8 ratio was calculated at each data point. The three ratios were averaged to yield a mean CD4/CD8 ratio per subject.) There were no missing data points. The specimens were obtained between 9:00 and 11:00 AM to control for circadian rhythm (22). PBL were isolated by density-gradient centrifugation (23, 24). We have previously reported no significant differences in percentages of CD2’ (P = 0.40) and CD4’ (P = 0.38) lymphocytes in a comparison of density-gradient separation and whole blood lysis, with a marginal difference in CD8’ cells (P = 0.04) (23). The fluorochrome-conjugated fluorescein isothiocyanate (FITC) or phycoerythrin (PE) monoclonal antibodies (MoAb) used in this study, with their major reactivities, are listed in Table 1 (25). The technical aspects of immunostaining have been reported previously (23. 24). PBL subsets values were obtained by analysis of dual-parameter histograms, each containing 10,000 cells. Isotypic negative control MoAbs, labeled with FITC, or PE, were used to determine the negativei positive cutoff point. Percentages of cells reactive (or nonreactive) with each pair of MoAbs were determined using the QUADSTAT program (Coulter Electronics). Percentages of MoAb positivity within the “lymphocyte” light scatter gate for each MoAb were adjusted based on the CD45 “bright” and CD14 positivity in order to compensate for erythrocytes, neutrophils, and monocyte contamination of this light scatter region (23). These corrected percentages were then used to determine the absolute number of MoAb-positive cell subsets by multiplying the total lymphocyte count obtained from a standard hematology profile by each corrected subset percentage.
TABLE 1 Monoclonal Antibodies
CD”
MoAbb
Main reactivity; other reactivity
Source’
CD2 CD4 CD5 CD8 CD14 CD16 CD20 CD21 CD45 CD57 NC.’
CCT 11 CC T4 Leu 1 cc T8 Leu M3 Leu 11 Leu 16 B2 HLe-1 Leu 7 HLA DR
T cells (SRBC receptor); NK cells Helper/inducer T cells T cells (strong); subset of B cells (weak) Suppressor/cytotoxic T cells; NK cells Monocytes; granulocytes Natural killer cells, granulocytes, monocytes B-cell restricted Mature B cells All leukocytes Natural killer cells; T-cell subset Class II major hi&compatibility antigen; B cells, monocytes, “activated” T cells and NK cells
c C CD C BD BD BD C BD BD BD
Fluorochrome”
__a Clusters of differentiation (CD) as defined by the Fourth International Workshop on Leukocyte Antigens, Vienna, 1989 (25). ’ Monoclonal antibody. c C, Coulter Immunology, Hialeah, FL; BD, Becton-Dickinson, Mountain View, CA. d P, phycoerythrin; F, Fluorescein isothiocyanate. e Not clustered.
P P P F P P F P F F F
LYMPHOCYTE
Data Analysis The first step in data analysis was to examine the normality of the population distribution of each immunophenotypic measure. For most measures, the normality assumption was not met, so nonparametric procedures based on ranks (if readily available) were used in subsequent comparisons. Second, analysis of covariante (ANCOVA) was used to adjust the data for agerelated group differences which occurred on a few selected measures. Third, immunophenotypic values from the RA and OA groups were compared using the Wilcoxon rank sum test. Fourth, disease activity measures were correlated with the immunophenotypic subsets using Spear-man correlation coefficients to determine if disease activity was related to any observed group differences. Fifth, the PBL subset values were compared for RA patients taking cytotoxic medications, RA patients taking noncytotoxic medications, and the OA patients (Kruskal-Wallis test). Sixth, the effect of prednisone on immunophenotypic subsets was analyzed using the Wilcoxon rank sum test. Due to the large number of statistical comparisons, the significance level was adjusted to 0.01. RESULTS
Age Comparisons
for RA and OA Patients
Since age differences the OA subjects (P = tion coefficients were tionships between age
239
SUBSETS IN RA
were found between the RA and 0.007), Spearman rank correlaused to examine possible relaand each of the immunophenoTABLE
typic measures. Age was found to be correlated with the total lymphocyte count and with absolute numbers of CD2’ and CD4+ lymphocytes. Age also correlated with relative percentages of lymphocytes positive for CD2+ and CD8+, as well as CD57+/CD16-, CD57-/ CD16’, and CD57+/CD16+ NK cells. Therefore, subsequent comparisons of these specific immunophenotypic measures were accomplished with ANCOVA using age as a covariate. Lymphocyte
Subsets of RA and OA Patients
The relative and absolute values for lymphocyte subsets of RA and OA patients are shown in Table 2. Statistically significant differences in percentages of cellular subsets between the two groups were found for CD5+/CD20+ B cells (P = 0.0002), CD20+/CD21B cells (P = O.OOOl>, CD57+/CD16lymphocytes (P = 0.003), and CD57+/CD16+ lymphocytes (P = 0.007). In all of these subsets, the magnitude of the observed difference was small, so the biological importance of each statistically significant difference is uncertain. The total lymphocyte count differed significantly between the groups (P = 0.0006; RA = 1309 cell+l; OA = 1598 cells/pi). Significant differences were found between patient groups in absolute numbers of CD2+ lymphocytes (P = 0.002), CD4+ lymphocytes (P = 0.0005), CD20+ B cells (P = 0.007), and CD20f/ CD21- B cells (P = 0.0001). Overall, the most notable numerical differences between the groups were observed in the decreased absolute numbers of CD4+ lymphocytes in the RA group, which in turn was re2
Comparison between RA and OA Patients
on Lymphocyte
RAb Immunophenotypic
subsets
Total lymphocyte count CD2 + lymphocytes’ CD4 + helper/inducer cells CD8 + suppressorkytotoxic cells CD4/CD8 ratid “Activated” CD2 + lymphocytes (CD2 +/HLA-DR+ Class II MHC antigen (HLA-DR) Total B cells (CD20) CD5 + /CD20 + B cells CD20+/C!D21- B cells CD20+/CD21+ B cells CD57+/CD16lymphocytes CD57-/CD16+ lymphocytes CD57+/CD16+ lymphocytes ; gr6y
of initial,
)
Subsets” OA’
P valued
Absolute number
Percent
Absolute number
Percent
Absolute number
1309 1062 670 283 3.6 50 185 96 21 23 74 351 139 82
80.4 51.6 21.4 3.6 4.1 14.5 7.4 1.6 1.8 5.6 26.3 11.6 6.6
1598 1284 829 296 3.4 46 206 132 17 57 75 327 161 75
80.4 52.0 18.6 3.4 3.1 13.2 8.1 1.0 3.5 4.6 20.6 10.5 4.8
0.0006* 0.002* 0.0005* 0.37 0.39 0.84 0.08 0.007 0.04 0.0001 0.79 0.78 0.13 0.89
Percent 0.99* 0.91 0.04* 0.39 0.05 0.36 0.19 0.0002 0.0001 0.12 0.003* 0.11* 0.007*
3-month, and 6-month measures for each subject expressed as a group mean.
‘N = 47: d Data not normally distributed, therefore Wilcoxon rank sum nonparametric e N = 46 for OA group due to unusable lab data for one patient. ‘Ratio of percentages of CD4 and CDS. * ANCOVA age-adjusted P values for group differences.
P values are reported, except for age-adjusted comparisons.
240
CALDWELL
fleeted in the decreased total number cytes. Correlation of Lymphocyte Disease Activity
of CD2 + lympho-
Subsets, and RA
Within the RA sample, Spear-man correlations were used to examine relationships between measures of RA disease activity and the absolute values of the PBL subsets where significant differences in absolute values were found between RA and OA patients (Table 3). Interestingly, there were no significant correlations between the disease activity measures (i.e., total active joint count, morning stiffness) and the PBL subsets for which group differences were found (i.e., total lymphocyte count, CD2+ lymphocytes, CD4+ lymphocytes, CD20+/CD21B cells). Thus, the observed PBL subset differences between the RA subjects and the OA subjects did not appear to be accounted for by variations in disease activity. Modulation of Lymphocyte Cytotoxic Therapy
Subsets by
To explore other possible reasons for differences in PBL subsets between the RA and the OA subjects the relationship between lymphocyte subsets and treatment with DMARDs was analyzed (Table 4). The hypothesis was that treatment of RA patients with cytotoxic agents might account for the lower PBL counts. Thus, the 63 RA patients taking DMARDs were divided into two groups. Medication group 1 (MGl) was composed of 42 RA patients receiving noncytotoxic treatment with gold (n = 271, penicillamine (n = lo), or hydroxychloroquine (n = 4). One additional MGl patient was taking hydroxychloroquine at baseline, hydroxychloroquine at the 3-month assessment, and gold at the 6-month assessment. Medication group 2 (MG2) was composed of 21 patients receiving cytotoxic treatment with methotrexate (n = 91, azathioprine (n = 91, or cyclophosphamide (n = 3). Two of the MG2 TABLE3 Correlation between Measures of RA Disease Activity and Absolute Numbers of Lymphocyte Subsets”
Total lymphocyte count TAJCb MS’
CD2 + lymphocytes
V3.M
- 0.04 -0.11
Note. All P values Averages of initial, measures. a Cells per microliter. b Total active joint ’ Morning stiffness
- 0.08 - 0.09
CD4+ helper/ inducer cells (CD4) 0.04 -0.12
patients were taking cyclophosphamide at baseline, methotrexate at the 3-month assessment, and cyclophosphamide at the g-month assessment. One MG2 patient was taking methotrexate at baseline, methotrexate at the 3-month assessment, and azathioprine at the 6-month assessment. The relative and absolute values for the lymphocyte subsets in MGl, MG2, and the OA group are shown in Table 4. Differences in the absolute counts between the RA and the OA groups were due primarily to the lower absolute lymphocyte counts in the subgroup of RA patients receiving cytotoxic medications (MG2). Specifically, MG2 differed significantly from the OA patients in absolute numbers of CD2’ lymphocytes, CD4+ lymphocytes, HLA-DR+ lymphocytes, CD20’ B cells, CD20+/CD21B cells, and CD20C/CD21 c B cells; whereas MGl differed from the OA patients only on absolute numbers of CD4’ lymphocytes, CD2O.Y CD21 - B cells, and CD20+/CD21+ B cells. Regarding percentages of PBLs, both MGl and MG2 differed significantly from the OA patients in CD5+/CD20t B cells, CD20+/CD21B cells, and CD57”/CD16lym. phocytes. Differences in disease activity between MGI and MG2 did not appear to account for the observed differences in PBL subsets because Wilcoxon rank sum tests revealed that MGl and MG2 did not differ in total active joint counts or estimates of morning stiffness. Effects of Prednisone
on Lymphocyte
Subsets
To more fully delineate the impact of medication on PBL subsets, the RA patients receiving low-dose prednisone (~10 mg/dayJ were analyzed separately within both MGl and MG2 (data not shown). There were 11 patients receiving prednisone in MGl and 9 patients receiving prednisone in MG2. The data revealed that there were no significant differences in lymphocyte subsets between those RA patients receiving low-dose prednisone and those not receiving prednisone. This observation suggested that chronic low-dose prednisone did not influence the lymphocyte subsets in the peripheral blood of patients with RA. It should be pointed out, however, that the statistical power of this analysis should be considered given the low number in each group. DISCUSSION
CD20+/CD21-B cells 0.19 - 0.20
for Spearman correlations are >O.lO. 3-month, and 6-month values were used for all
count. (hours).
ET AL.
Because RA is an autoimmune disease, it is rational to consider the possibility that alterations of immune cellular subsets might be valuable from an accessible compartment such as the blood and possibly provide useful information related to immune status or disease activity. Clearly, most of the pathology, and probably the traffiking of lymphoid cells, is aimed at the inflamed synovium. However, if lymphoid cells in peripheral blood are a direct (or indirect) reflection of disease activity, then evaluation of this easily accessible com-
LYMPHOCYTE
Comparisons among RA Medication
TABLE 4 Subgroups and OA Group on Lymphocyte Subsets”
RA MGlb Immunophenotypic
subsets
Total lymphocyte count CD2+ lymphocytes Helper/inducer cells (CD41 Suppressor/cytotoxic cells (CDS) CD4/CD8 ratio’ “Activated” CD2+ lymphocytes (CD2+/HLA-DR+) Class II MHC antigen (HLA-DR) Total B cells (CD201 (from CD20f/CD21+) CD5 + /CD20+ B cells CD20’/CD21B cells CD20+/CD21+ B cells CD57+/CD16lymphocytes CD57-/CD16+ lymphocytes CD57+/CD1GC lymphocytes
241
SUBSETS IN RA
RA MG2’
Absolute number
%
1448 1160 746 293 3.6 55 214 115 23 27 87 373 151 82
80.0 51.6 20.5 3.6 4.0 14.8 7.8 1.6 1.8 6.0 26.2 11.4 6.2
Absolute number 1031 866 524 262 3.8 40 125 59 17 13 46 307 117 81
OAd % 81.2 51.8 23.2 3.8 4.2 13.9 6.5 1.6 1.7 4.8 26.7 12.0 7.2
Absolute number 1598 1284 829 296 3.4 46 206 132 18 57 75 327 161 75
P
% 8;4 52.0 18.6 3.4 3.1 13.2 8.1 1.0 3.5 4.6 20.6 10.5 4.8
valuee
Absolute number
%
0.0001*~~ 0.0005*f6
0.86*
O.OOO1*f~~h
0.99
0.30 0.66 0.04
0.26 0.66 0.13 0.32
0.0004f~ 0.0002fg
0.09
0.05 0.0001~~~ O.OOwh 0.14 0.02 0.41
0.0007g*h 0.0001~~~ 0.05 O.OlM,h 0.27* 0.02*
a Averages of initial, 3-month, and 6-month measures for each subject expressed as a group mean. b RA patients taking gold, penicillamine, or hydroxchloroquine (N = 42). ’ RA patients taking methotrexate, azathioprine, or cyclophosphamide (N = 21). d OA patients taking nonsteroidal anti-inflammatory drugs (N = 47). e Data not normally distributed, therefore nonparametric Kruskal-Wallis P values are reported, except for age-adjusted comparisons. The P values represent comparisons of all three groups. f Post-hoc comparisons revealed RA MGl significantly different from RA MG2. 8 Post-hoc comparisons revealed RA MG2 significantly different from OA group. h Post-hoc comparisons revealed RA MGl significantly different from OA group. ’ Ratios of percentages of CD4 and CD8 were calculated for each subject and then averaged over the three time points to yield a mean ratio per subject. These numbers were then averaged over the entire group to yield the mean CD4XD8 ratio reported above. *ANCOVA age-adjusted P values for group differences.
partment might be useful. It is not clear that alterations in lymphocyte subsets reported in earlier studies reflect disease activity or if they are secondary to medications or other intervening factors. In this study, we examined peripheral blood lymphocyte subsets from a large group of patients with RA or OA. Patients with RA had lower absolute numbers of total lymphocytes, total CD2+ lymphocytes, CD4+ lymphocytes, total CD20f B cells, and CDZO+/CDZlB cells (all P < 0.002). Differences in total lymphocytes and total CD2+ lymphocytes were associated with cytotoxic drug therapy used by some of the RA patients (Table 4). Interestingly, there were no significant correlations between PBL subsets and either RA disease activity or the use of low-dose prednisone within the RA groups. These results suggested that chronic therapy with DMARDs in RA may be associated with PBL subset abnormalities independent of RA disease activity. Statistically significant differences were also found between the RA and OA groups in some of the quantitatively smaller lymphocyte subsets. Several investigators have reported significant differences between cellular subsets of patients with active or inactive RA (3,4,6,7,9, lo), but the differences between “active” and “inactive” categories were minimal (or not reported) in some studies and significant interstudy variability in classification was present.
These incongruities, coupled with large within-subject variability in lymphocyte subset values, make clinically useful interpretations of the data difficult. Additionally, medications are known to alter lymphocyte subsets and thus may have obscured real differences in the distribution of lymphocyte subsets related to disease activity. To overcome these problems, comprehensive assessments of disease activity were performed, and statistical analyses were used to evaluate known or suspected intervening factors, including medications. In this study, there were no statistically significant correlations between disease activity measures and the PBL subset values on which group differences were found. However, differences in subset values between RA and OA patients were obvious and thus required further analysis of medication effects. Many patients with RA receive cytotoxic medications which have been shown to produce lymphocytopenia (26-30). Weekly pulse-dose methotrexate therapy in patients with RA has been shown to produce a modest decrease in the total lymphocyte count after only 13 weeks of therapy, but with relative increases in the percentage of total T cells and CD4+ lymphocytes (26). In another short-term study on the efficacy of methotrexate in RA, no significant differences in the PBL subsets were found in 12 weeks (27). However, a longterm study (2 years) revealed increased percentages of
242
CALDWELL
total T cells (CD3) and T-helper cells (CD4) compared to baseline values (281, which is consistent with the 13-week study (26). Cytotoxic therapy with azathioprine and cyclophosphamide also has been associated with lower total lymphocyte counts, but not to changes in the relative percentages or absolute numbers of T or B cells (29, 30). Our study, in which 94% of the RA patients had been treated with stable doses of cytotoxic therapy for at least 6 months, produced statistically significant differences in the total lymphocyte count. Second, significant differences were observed in absolute numbers of the CD2+ T cells, CD4+ T-helper cells, CD20+ B cells, and HLA-DR+ lymphocytes, but not in percentages of lymphocytes when the cytotoxic and noncytotoxic groups of RA patients were compared. This is consistent with earlier studies in which a “control” group of RA patients treated with NSAIDs, prednisone, or gold was investigated (29, 30). These patients had significantly higher total lymphocyte counts than patients taking cytotoxic agents, but were still below that of normal controls. These findings are in accord with the current study of RA patients in each medication group and OA controls. Beyond these medication-related findings, both groups of RA patients differed from the OA controls in other subsets. Corticosteroid therapy, which is also known to induce lymphocytopenia (31331, is commonly used in conjunction with other DMARDs. Therefore, sustained modulation of cellular subsets by the chronic use of these agents in diseases of abnormal immune regulation is of interest. In low oral doses, corticosteroids appear to produce only transient lymphocytopenia in normal subjects, with CD4+ T-helper cells an CD2+ T cells affected to a much greater extent that CD8+ T-suppressor cells (31). Similar transient responses to prednisone (lo-mg dose) have been found in a small group of patients with RA, although CD3, rather than CD2, was used to measure T cells (32). The use of lowdose prednisone in our study was found not to contribute to subset changes, but this finding may be due to the fact that prednisone was used long term in the present study as compared to short-term usage in other studies (31-33). Male OA patients receiving stable doses of NSAIDs were selected as the control group for this study. Comparison of the PBL subsets from the control patients in this study with the data from control subjects from other studies revealed comparable values (4-6). Thus, the findings from this study did not appear to be attributable to characteristics of the OA control sample. The four cellular subsets (CD5+/CD20+ B cells, CD20+/CD21B cells, CD57+/CD16’ NK cells, and CD57+/CD16lymphocytes) that were found to differ in percentage from OA controls in both the cytotoxic and noncytotoxic groups of RA are of interest. Since the same alterations occurred in both groups of RA pa-
ET AL.
tients, consideration must be given to the possibility that these changes are disease related. For instance, CD5 +/CD20+ B cells are known to be increased in RA and have been shown to be committed to production of IgM with rheumatoid factor activity (34, 35). The percentages of CD5 + B cells in the present study are consistent with those previously reported (35). However, in the present study, the percentage of CD20C/CD21 B cells was decreased in the RA patients. This contrasts with studies which show a reduction of CD21 from normal B cells following in vitro B-cell activation and an increase in CD20+/CD21B cells in diseases characterized by B-cell activation and hypergammaglobulinemia (36, 37). Since B cells account for only 78% of the total lymphocytes, the significance of these findings is uncertain. It should also be noted that somewhat different numbers of total B cells were measured when using the CD20-containing MoAb pair Bl/B2 compared to Leu lG/Leu 1. However, the percentage of CD20’ cells should be the same with either MoAb. The reason(s) for such differences is not entirely clear. Data from this laboratory demonstrate that while these two MoAb bind to the same antigen on human B cells, cells from squirrel monkeys bind only Bl, suggesting a difference in epitopic-binding sites (unpublished data). The differences found in the present study were small, ranging from 0.6 to 2.2%, were not statistically significant (P > 0.051, and were found to correlate well (Pearson coefficient, 0.88 for absolute CD20). Thus, the minor discrepancy in number of total B cells measured may be related to reagents used, experimental conditions, or possibly epitopic differences in some individuals. The differences in the lymphocyte subsets defined by CD57 and CD16 reactivity are also not well explained (38). Since CD57 is present on a subgroup of CD2+ T cells as well as NK cells, the CD57+/CD16subgroup may include this subset of T cells accounting for some of the differences. It should be understood that RA is a heterogeneous disorder with variable immunologic dysfunction, suggesting that differences in smaller subsets may fall within the spectrum of immune abnormalities in RA. In conclusion, this study revealed that most, but not all, of the changes in PBL subset values found in RA patients were probably the result of therapy utilizing DMARDs. There were no significant correlations between PBL subsets and RA disease activity, thus suggesting that chronic therapy with DMARDs in RA may be associated with PBL subset abnormalities independent of the disease activity of RA, but still related to the presence of the disease. A variety of differences were found among the smaller lymphocyte subsets which may be disease related; however, the clinical significance of these findings is unclear. Conflicting results from previous studies may have resulted from methodological differences or failure to account for rel-
LYMPHOCYTE
evant patient characteristics logical effects.
or important
SUBSETS
pharmaco-
ACKNOWLEDGMENTS
The authors gratefully acknowledge the contributions of Barb Cullen, Thomas Feldbush, Kristofer J. Hagglund, Robert Hoffman, Frank O’Sullivan, Gordon Sharp, and Elaine Smith.
15. 16.
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