Antilymphocyte antibodies in rheumatoid arthritis: Increased reactivity against activated lymphocytes

CI.INICAI.

IMMUNOLOGY

AND

IMMUNOPATHOI

OG\r

20. 246-254

11981)

Antilymphocyte Antibodies in Rheumatoid Arthritis: Reactivity against Activated Lymphocytes

Increased

Sera from patients with rheumatoid arthritis (RA) were tested for cold reactive lymphocytotoxic antilymphocyte antibodies (ALA). Of 25 sera. 3 had reactivity with resting lymphocytes, 7 of 25 sera reacted with cultured and PWM-activated lymphocytes, and 9 of 25 sera had reactivity with Con A-activated lymphocytes. Positive sera had minimal increases in ALA titer with cultured cells and marked increases with mitogen-activated target cells. ALA against activated lymphocytes were found to be predominantly IgM cold reactive. complement-dependent antibodies. Increased ALA reactivity was directed against lymphoblasts which were purified on PVP-coated colloidal silica (PercoIl) gradients. RA sera reactivity against lymphoblasts was also increased using an indirect immunofluorescent technique. ALA activity against activated lymphocytes was absorbed with fresh mononuclear cells.

INTRODUCTION

Naturally occurring antilymphocyte antibodies (ALA) have been demonstrated in a variety of conditions including systemic lupus erythematosus (SLE) (1, 2), rheumatoid arthritis (RA) (1, 2). viral illness (3), after vaccination (4), and rheumatic fever (5). In addition, ALA are present in the consanguinous and nonconsanguinous relatives of SLE patients (6-8). Close household contacts of SLE patients were found to have a higher prevalence of ALA than nonhousehold contacts. Laboratory personnel involved in SLE research also have an increased incidence of ALA when compared to normals (9). This association of ALA with autoimmune diseases, postinfectious states, asymptomatic relatives of patients with autoimmune disease, and laboratory personnel has prompted speculation that ALA may represent a marker of an environmental agent operative in these conditions. This agent may be viral in nature but numerous studies have thus far failed to identify a specific organism ( 10, 1I ). Recently, ALA reactivity has been associated with different subtypes of lymphocytes (12- 16). We were interested in whether the state of activation was involved in this mechanism. Lymphocytes activated by concanavalin A (Con A) have increased in HLA (17), Ia ( 18, 19), and DRw (20) reactivity. Because ALA also have surface membrane specificity. there may be a correlation with these phenomena. A study was initiated to investigate ALA reactivity with mitogen activation of normal lymphocyte targets. RA sera were selected as the source of 1 Recipient of National Institute of Health ? Present address: Department of Medicine.

Clinical Investigator Award University of Minnesota, ‘46

0090- 122918 l/080246-09$0

1.00/O

Cupynghf 8 1981 by Academic Press. Inc. 411 rights of reproduction in any form recerved.

lK08 AM00793-01. Minneapolis, Minn.

jj455.

RHEUMATOID

ARTHRITIS

ANTILYMPHOCYTE

ANTIBODIES

247

ALA because ALA activity against fresh lymphocytes has been previously found in low titers (21). Investigations of ALA against activated lymphocyte targets may give insight into ALA antigens on the surfaces of activated lymphocytes and may be a more sensitive test for ALA in this disease. MATERIALS

AND METHODS

Subjects. Thirty-one serum samples were selected from 24 patients with RA as determined by the American Rheumatism Association criteria (22). These were divided into two groups: Group I included 18 patients with either Stage I or II disease (23) and with varying degrees of clinical activity. Medications in these 18 patients included nonsteroidal anti-inflammatory drugs, aspirin, gold, and hydroxychloroquine. None of these patients were on steroids, penicillamine, or immunosuppressive agents. Group II included 6 patients with more severe RA (Stage II or III) who were chosen for ALA positivity in the indirect immunofluorescent studies of purified lymphoblasts. Medications of Group II patients included prednisone, penicillamine, and/or hydroxychloroquin. Serum samples from 12 individuals matched for age and sex with the RA patients were also studied as controls. Lymphocytes. Lymphocytes were separated from heparinized whole blood of normal human donors by Ficoll-Hypaque gradient centrifugation (24). Monocyte depletion was effected through glass adherence on petri dishes at 37°C for 30 min. Peroxidase-positive cells using benzidine dihydrochloride stain (25) were generally 2% or less in monocyte-depleted preparations. Target lymphocytes. Target cells for ALA determinations were prepared as resting, cultured, and activated lymphocytes. Resting cells were Ficoll-separated peripheral blood mononuclear cells (MNC) or monocyte-depleted lymphocytes as described above. Cultured cells were lymphocytes incubated in RPM1 1640 with 20% fetal calf serum (FCS) for 48 hr. Activated cells were lymphocytes incubated for 48 hr in RPM1 1640 with 20% FCS and either Con A (20 ,wg) or pokeweed (PWM) (40 pg) mitogens. Concentration of mitogens and duration of incubation were selected on optimal [3H]thymidine uptake. Cell numbers, percentage blasts as determined by numbers of large cells, and cell viability by trypan blue exclusion were recorded after all incubations. Cell viability was 98% or greater after all cultured procedures. Antilymphocyte antibodies. ALA were determined both by the two-step microcytotoxicity method of Terasaki (26) and indirect immunofluorescence of suspensions of lymphocytes (27). In the microcytotoxicity procedure, target cells were prepared as resting, cultured, and activated lymphocytes as described above. Rabbit complement (Pel Freeze, Rogers, Ark.) incubation was for 3 hr at 15°C. Dilutions of RA sera were made with RPM1 1640 and 20% human ABpositive serum. ALA positivity was determined to be 30% or greater dead cells per 100 counted. Normal human serum and RPM1 AB-positive diluent controls had < 10% dead cells in each experiment. Percentage dead cells for controls tended to be lower for resting target cells and higher for the cultured or activated target cells but were always ~10%. ALA activity was also expressed as cytotoxicity index (CI) which was calculated by dividing the sum of all cytotoxic reactions by the

248

SEARLES.

MESSNER,

AND

HERMANSON

number of donors tested. Positive CI was determined to be greater than 0.20 as all control sera except one (see Results) fell well below that level. ALA were also determined using indirect immunofluorescence of suspensions of lymphocytes as previously described (27). Briefly. l-2 x 10” monocytedepleted resting or mitogen-activated lymphocytes were incubated serially with test serum and with fluorescein-labeled (FIT0 goat anti-human IgM or 1gG (Meloy, Springfield, Va.) for 1 hr at 4°C. In each test 300 cells were counted and results were recorded as the total number of fluorescent cells per 100 counted. Cell viability tested by trypan blue exclusion was 9%; or greater before and after ALA assay. Pew011 gwtfknts. Lymphoblasts were separated on discontinuous gradients of polyvinylpyrrolidone (PVP)-coated colloidal silica (Percoll). (Pharmacia Fine Chemicals, Piscataway. N.J.) (28). Percoll was made isotonic with 1.5 M phosphate-buffered saline (PBS) and is referred to as 100% solution. The 100% sobtion was then serially diluted with 0.15 M PBS to 60, 50, and 20% solutions. TWOmilliliter volumes were sequentially layered over the 100% layer containing suspensions of monocyte-depleted lymphocytes cultured for 4- 5 days with and without mitogens and centrifuged at 1500 rpm for 15 min at room temperature. The cells in the 50-20% interphase were 80% or greater lymphoblasts as determined by size (light microscopy) and morphology of Wright’s stained cytocentrifuged preparations. Functional characteristics of Percoll-concentrated lymphocyte functions were evaluated by [3H]thymidine incorporation and the 50--20% band had greatest uptake in 48 hr. Ahsorptions. Sera from RA patients were absorbed with equal volumes of Ficoll-separated MNC. Each serum was absorbed three times for 1 hr at 4°C. Cell viability by trypan blue exclusion was ~-95% after absorption. Sera were tested for ALA prior to and after absorption. RESULTS

In the initial ALA screen, 25 sera from 18 RA patients in Group 1 were tested for ALA against target lymphocytes from 7 normal donors of varying HLA types (Table 1). Target lymphocytes had different average number of percentage blasts present in the final preparations: resting (O%), cultured (4%). PWM activated (27%), and Con A activated (40%). Of 25 sera, 3 had reactivity against the resting lymphocytes. When the lymphocytes were cultured for 48 hr in RPM1 1640 with 20% FCS. without mitogen or with PWM mitogen, 7 of 25 serum samples had increased ALA activity (CI > 0.20). When the target lymphocytes were cultured in RPM1 1640 with 20% FCS and Con A mitogen, 9 of 25 sera had CI > 0.20. Analysis of cytotoxic ALA patient by patient showed 2118 patients were reactive with resting lymphocytes, 5/18 with cultured and PWM-activated lymphocytes. and 6/18 with Con A-activated lymphocytes. Of 6 patients tested with multiple serum samples, 3 had conflicting ALA activity in different samples. These samples were drawn at different times and at various levels of clinical activity. All 12 control sera had CI < 0.05 with resting, cultured, and activated lymphocytes except for one serum. This sample was obtained from an immunology research technician who was positive with Con A-activated targets only (CI = 0.50). These data are consistent with previous reports of increased ALA activity in some laboratory technicians (9).

RHEUMATOID

ARTHRITIS TABLE

ALA

IN 25 UNDILUTED

RESTING,

CULTURED,

SERUM

SAMPLES

PWM-ACTIVATED,

OF

ANTILYMPHOCYTE 1 18 RA AND

PATIENTS

249

ANTIBODIES

FROM

GROUP

CON-A-ACTIVATED

I AGAINST

LYMPHOCYTES

FROM 7 NORMAL DONORS Activated RA Serum 101 102 103 104A B 105A B 106A B 107 108 109 110 1llA B ll2A B 113 ll4A B C 115 116 117 118

Resting

Cultured

0” 0 0 0.04 0.05 0.50 0.39 0 0 0 0 0 0 0 0.44 0 0 0 0 0 0 0 0 0 0

0.24 0 0.07 0.22 0.39 0.70 0.25 0.24 0 0 0 0 0.06 0.19 0.41 0.03 0 0.13 0 0.12 0 0 0.05 0.05 0

PWM 0.14 0 0 0.04 0.39 0.64 0.27 0.13 0 0 0 0 0 0.24 0.56 0.26 0.07 0.29 0.04 0 0 0.07 0.07 0 0

Con A 0.27 0 0 0.31 0.46 0.63 0.41 0.26 0 0.07 0.13 0.04 0.07 0.26 0.73 0.26 0.07 0.04 0 0 0 0.11 0.13 0 0

n Results are expressed as cytotoxicity index (CI) which was calculated by dividing the sum of all cytotoxic reactions by the number of donors tested.

The survey data in Table 1 suggest similar increases in ALA activity can occur with the cultured and activated lymphocyte targets. However, marked increases in ALA titer were usually found with Con A-activated lymphocytes while slight or no increases in titer were found with the cultured cells. Titration of two ALA positive against resting, cultured, and Con A-activated target cells from different donors is shown in Table 2. When RA serum 105 was tested against Donor A, the ALA titers were I:64 against resting cells, 1:4 against cultured cells, and 1:4000 against Con A-activated cells. When RA serum 105 was tested against Donor C, the ALA titers were I:32 against resting cells, 1:256 against cultured, and 2 1:8000 against Con A-activated target cells. Titer of ALA activity had greater increases against the Con A-activated targets. These data are not reflected in Table 1 but have been consistently evident throughout our experiments. Donor variability previously described with cytotoxic ALA against resting cells was also present against the cultured and activated targets (note ALA titers when RA 105 was tested against Donor B were less than against donors A and C)..

250

SEARLES,

MESSNER,

AND

TABLE TIMERS

OF ALA

ACTIVITY

CON

Serum

A-ATTIVATED

IN RA

2 4ciAiNsr

SERB

CELLS

HERMANSO&

FROM

RESIINCI.

DIF ERENT

C~,I

FL~R~I).

XVI)

DONORS

Con A Activated

Dunol

Resting

Cultured

RA 105

.4 B c

1:64” I:4 I:32

I:4 I: I6 I:256

I :4000 I:72 - I : 8000

RA

A B D

I:64 Neg I:32

1:x I: I6 I:128

! : 2000 I:16 i:lO2-1

III

” Results are titers of ALA with positivity determined Dilutions of sera made with RPM1 + 20% AB-positive mal human serum were IOQ or less dead cells.

as 3W serum.

or greater Negative

dead cells per 100 counted. controls of diluent and nor-

Additional experiments demonstrated the ALA system with activated lymphocytes is dependent on complement, time. and temperature. None of the sera tested were cytotoxic unless rabbit complement was added to the cultures. Temperature was evaluated by culturing RA sera at 15 and 22°C. Both cold and warm reactivity were present but reactivity was consistently greater at 15°C. Both types of reactivity increased after Con A activation. Terasaki (26) has shown the optimal incubation time with complement in the 15°C microcytotoxicity procedure is 2 hr. Duration of incubation with rabbit complement was evaluated at 15°C with ALA tested against Con A-activated lymphocyte targets. Increasing amounts of cytotoxic ALA activity were found with longer incubation periods. However, incubation of greater than 3 hr resulted in increased background cytotoxicity without a significant increase in sensitivity. Thus the 3-hr incubation period at 15°C with rabbit complement also appears optimal for the Con A-activated cells. Mitogen-activated lymphocytes contained heterogenous populations of stimulated blast cells and stimulated nonblast cells. To isolate the lymphoblasts. discontinuous gradients of PVP-coated colloidal silica (Percoll) were used as noted above. In a typical experiment four ALA-positive RA patients from Group II were evaluated against unseparated mitogen-activated lymphocytes and Percollseparated lymphoblasts from the 50-20% interphase (Table 3). ALA activity against the unseparated activated lymphocytes was found to be concentrated in the lymphoblast cell layer, while minimal ALA activity was directed against the nonblast layer. Further studies using cells cultured without mitogen showed no concentration of lymphoblasts at the 50-20% interphase (data not shown). ALA were also evaluated by indirect immunofluorescence of suspensions of Percoll-separated lymphoblasts. A representative experiment is demonstrated in Table 4 where three ALA-positive RA sera from Group II were tested and IgM ALA activity was detected against 31 to 70% of lymphoblasts and IgG ALA activity against 7 to 17% of lymphoblasts. The IgG ALA reactions against lymphoblasts in RA patients were similar to the NHS while IgM ALA were increased over NHS in all three patients. A slight increase in IgG ALA was noted in RA 122.

RHEUMATOID

ARTHRITIS

ANTILYMPHOCYTE

TABLE ALA

CYTOTOXK AND

IN

RA

PATIENTS

PERCOLL-SEPARATED

3

UNSEPARATED

LYMPHOBLASTS

AND

CON

119 120 121 122

A-ACTIVATED

NONLYMPHOBLASTS

LYMPHOCYTES, AS TARGET

CELLS

Percoll cell fractions

Unseparated Con Aactivated lymphocytes

Serum RA RA RA RA

USING

251

ANTIBODIES

50-20%”

70” 80 60 70

60-M%

80 80 90 60

20 10 10 10

” 50-20% band contained 80% or greater lymphoblasts: 60-50% band contained 10% or less lymphoblasts. ’ Percentage dead cells. Cytotoxicity assay performed with antibody incubation at 4°C for 1 hr and complement at 22°C for 2 hr.

These data also show that increased ALA activity against activated cells can be demonstrated by the suspension method and is not only a function of the cytotoxicity procedure. Other experiments utilizing 2-mercaptoethanol reduction of RA sera (29) also demonstrated that the ALA class was predominantly IgM. ALA in RA patients were quantitatively compared to the known heteroantisera directed at lymphocyte surface antigens (Table 5). Anti-HLA-A,, anti-/3-2-microglobulin (anti-&p) (a gift from Dr. S. Ferrone, Scripps, Calif.) and anti-Ia (a gift from Dr. R. C. Williams, University of New Mexico) were chosen for investigation. Anti-HLA-A, activity was tested against an HLA-AZ-positive donor. The data indicate only slight increases against Con A-activated lymphocytes (one or two tube dilutions) with the more specific anti-HLA-A, and anti-P+. Greater differences were seen with anti-Ia as the negative activity found against resting lymphocytes increased to I:32 against mitogen-activated lymphocytes. The greatest increase in ALA titer was again found with the RA sera. We next looked at whether ALA antigens on activated lymphocytes were also present on the unstimulated cells. RA sera were absorbed with fresh Ficollseparated peripheral blood mononuclear cells (MNC) and tested for ALA activity against resting, cultured, and mitogen-activated lymphocyte targets. The data demonstrate resting MNC absorbed all ALA activity against all target cells inALA

ANTIBODY

CLASS

IN RA CON

TABLE PATIENTS

A-ACTIVATED

4 AGAINST

RESTING

LYMPHOCYTES

kM Sera PBS NHS RA 122 RA 123 RA 124

Resting 2 7 7 42 8

AND

LYMPHOBLASTS”

II@ Activated 1 10 31 70 27

Resting 1 3 6 12 5

I( ALA, Percentage positive cells using indirect immunofluorescence separated lymphoblasts.

Activated 1 4 17 11 7 of suspensions of Percoll-

252

SEARLES,

MESSNER, TABLE

AND

HERMANSON

5

COMPARISON OF ALA USING RESTING. CULTURED, RND Co& A-ACIIV~T~D L~MPHOTYTES THE SAME DONOR 4s TARGETS FOR HETEROANTISERA AND SERA FROM RA PATIENTS Serum

Resting

Heteroantisera Anti-HLA-A, Anti+‘,-p Anti-la Rheumatoid RA IO.5 RA Ill

Cultured

FROM

Activated

114” 1: 1024 Neg

I:4 1: 2048 NV

i. 16 1:204x I,32

I:64 I:64

I:4 I:8

1: 4000 1:2000

arthritis

” Results are titers of ALA with positivity determined Dilutions of sera made with RPM1 + 20% AB-positive human serum were 10% or less dead cells.

as 30%’ or greater dead cells per 100 counted. serum. Negative controls of diluent and normal

eluding Con A- and PWM-activated lymphocytes in all sera studied. These results suggest ALA antigens present on the activated lymphocytes were also present on the resting cells. DISCUSSION

These data confirm the presence of low-titer ALA activity in the sera of RA patients using resting lymphocytes as target cells. This activity is enhanced when cultured or mitogen-stimulated lymphocytes were used as targets. Of 25 sera, 3 had minimal sensitivity against resting lymphocytes from seven normal donors. When the target cells were cultured with Con A mitogen, 9 of 25 RA sera had ALA activity. ALA activity against the mitogen-activated targets was consistently higher than with the cultured target cells without mitogen and the Con A-activated cells were more sensitive than PWM-activated cells. Increased ALA activity with activated cells was limited to certain sera indicating the phenomenon is not present in all RA patients. Experiments also demonstrated that ALA against activated lymphocytes are IgM cold reactive, complement-dependent antibodies. Several explanations for these findings are worth consideration. First the increased ALA activity against activated cells could represent the inability of activated cells to repair membrane damage. In comparison to RA sera, antisera specific for certain MHC antigens (HLA-A2 and &,u) did not have the large increases in antibody titer against the activated target cells. The minor increases found in HLA-A, antibody titer could represent increased surface area as previously suggested using mitogen- and EB virus-activated lymphocytes (17). Therefore, although increased susceptibility to lysis of activated cells may be a factor, it is insufficient to explain the magnitude of differences seen with the RA sera. Increased reactivity of RA sera against activated cells was also noted using indirect immunofluorescence, a technique which is not dependent on cellular metabolism and cell lysis. The immunofluorescence data also suggest that cell fragility of activated cells is not a factor in our experiments. Another consideration is that ALA determinants on resting and activated lymphocytes are similar but have increased expression on the mitogen-activated cells.

RHEUMATOID

ARTHRITIS

ANTILYMPHOCYTE

ANTIBODIES

253

Alternatively, the determinants may be different through a modulating effect occurring during the activation process which alters their antigenicity or expresses an entirely new antigen on activated cells. It was therefore critical to differentiate whether ALA RA activity on the activated cells recognizes the same antigens derived from precursor cells or detects new antigens generated during the activation process. Our data demonstrated resting MNC absorbed all ALA activity against resting and activated targets suggesting the antigen is not unique to the activated state. In addition, our data and others have shown Ia determinants in activated states have increased expression (18, 19). The latter fact provides an important control for this phenomenon and suggests the possibility that the RA sera may be detecting an increased expression of Ia on activated cells. Finally, the increased ALA activity against mitogen-activated lymphocyte targets may represent an increased sensitivity of the surviving cells only and not the entire population. When lymphocytes are cultured with or without Con A or PWM mitogens over a 4%hr period, large numbers of the original resting cells do not survive. The precursor cells of the surviving lymphoblasts could represent an ALA sensitive subset of the original resting cells. Therefore the same RA sera positive for ALA against activated cells (CI > 0.20) would be defined as negative in the usual cytotoxic assay which uses the entire population of resting cells (CI < 0.20). After activation, the proportion of precursor cells in the surviving population would be greater and thus the ALA activity may now be termed positive. This possibility is supported by our data which shows that the greatest ALA activity is directed against the surviving lymphoblasts isolated on Percoll gradients. Reactivity with subtypes of lymphocytes has been demonstrated with juvenile rheumatoid arthritis (30) and systemic lupus erythematosus sera (12- 16). Our data show that RA sera have increased reactivity with mitogen-activated cells which could represent subclass specific antibodies directed at antigens enriched in activated lymphocytes. Further work is now in progress to more thoroughly characterize ALA activity of RA sera using purified lymphoblast cell populations. ACKNOWLEDGMENTS Supported in part by Career Development Award Grant X04 AM70301, Grant 2 ROl AMl3789-08 from NIH. and grants from National and New Mexico Arthritis Foundations.

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MESSNER.

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HERMANSOIV

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