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Immunologic reactivity in rheumatoid arthritis response to mitogens.

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Immunologic Reactivity in Rheumatoid Arthritis
Response to Mitogens
E. M. Lance and Stella C.Knight
The in vitro responsiveness of peripheral blood lymphocytes to a variety
of mitogens was compared in cells from normal volunteers, from patients
with rheumatoid arthritis and from patients with degenerative joint disease (osteoarthritis). A consistent and marked depression in the proliferative response to Conconavalin A was noted, while statistically comparable results were obtained with PHA and allogeneic lymphocytes. The
significance of these observations is discussed with particular reference
to-a possible discrete defect in a subpopulation of T cells in these
patients.
The most outstanding clinical feature of
rheumatoid arthritis is the formation and
persistence of chronic granulomata within
the joints and other tissues (1). This evidence for an ongoing immune response has
prompted a largely unrewarding search for
the antigen presumed to act as a trigger (2).
Another approach to mechanisms might be
to determine whether chronicity (regardless
of initial stimulus) is due to aberrant immune reactivity, ie, whether patients develop rheumatoid arthritis not because they
are exposed to a peculiar stimulus but because their response is unusual. If defects
in immune performance do contribute to
the pathogenesis of rheumatoid arthritis,
they might take the form of either an ininadequate response (immune deficiency) or
of inappropriate intensity (hyperresponsiveness).
T h e observations reported herein form
From the Division of Surgical Sciences, Clinical
Research Centre, Watford Road, Harrow, Middlesex, England.
Address reprint requests to E M Lance.
Submitted for publication January 24, 1974; accepted April 16, 1974.
part of a larger survey of overall immune
function in patients with rheumatoid arthritis. They suggest a deficiency in some
parameters of lymphoid function as compared to appropriate controls.
MATERIALS AND METHODS
Patients
Forty-four adult patients with definite or classic
rheumatoid arthritis, according to criteria of the
American Rheumatism Association (S), were studied.
Most were female (41), their ages ranged from 25
to 73 years, and most were receiving salicylates or
modest doses of antiinflammatory drugs (phenylbutazone or indomethacin). Approximately onethird were receiving maintenance gold salts, and a
few were maintained on low doses of steroids (less
than 8 mg of Prednisone per day). None was receiving cytotoxic or other immunosuppressive drug
at the time of study.
Control groups consisted of 17 patients with degenerative joint disease (osteoarthritis). and 17 normal volunteers. T h e former group was mostly female (12). while the sex distribution in the latter
group was approximately equal. Patients with osteoarthritis ranged between 40 and 80 years of age,
and many were receiving salicylates and antiinflammatory drugs. None was taking steroids or other
immunosuppressive drug.
Arthritis and Rheumatism, Vol. 17, No. 5 (September-October 1974)
513
LANCE ET AL
Normal volunteers were between 18 to 65 years
of age, and none was taking medicaments of any
kind at the time of study.
Evaluation of Lymphocyte
Responsiveness
1
10
9 .
a cpmx1~-3
7 -
6 5 -
Triplicate cultures containing 2 X 1oJ lymphocytes from defibrinated gelatin-sedimented peripheral blood were set up in 0.2 ml Dulbecco's medium in microplates (96 flat-bottomed wells, Cooke
Engineering). Autochthonous or pooled allogenic
serum/gelatine (0.25 ml per culture) was added.
The mitogens used were phytohaemagglutinin
(PHA: 0.01 ml of a 1:6 dilution, Burroughs Wellcome MR-lo), Concanavalin A (Con A; 4 pg, Calbiochem) or pokeweed mitogen (PWM: 4 pg, Difco).
These were the concentrations found to give optimal responses with cells from normal volunteers in
the presence of autochthonous or allogeneic serum
over the 3- to 4-day culture period. Cells from a
lymphoid cell line CLA-4 (derived from an infectious mononucleosis patient by Dr. Michael Steele)
were treated with mitomycin C (Nutritional Biochemicals, 25 pg/ml for 20 minutes at 37"C),
washed three times, and 0.025 ml of cell suspension
containing 2 X 10" cells per milliliter of Dulbecco's
medium were added to some cultures to assess the
response to histocompatibility antigens (MLC) (4).
H8-Thymidine (0.2 pCi, specificity activity 150. Ci
per mMole) was added 24 hours before harvesting
on day 4 (PHA, Con A and PWM) or day 6 (CLA4 stimulation) of culture. Processing the cultures
was similar to that previously described (5) except
that saline, trichloracetic acid and methanol washes
were performed by centrifuging the plate and discarding the supernatants. Finally, 0.2 ml of normal
sodium hydroxide was added to each well, and 0.1
ml of this transferred to scintillant. Results are
expressed in counts per minute, and the counting
efficiency was approximately 250/,.
The total lymphocyte counts in peripheral blood
of patients with rheumatoid arthritis were not
significantly different from those found in patients
with osteoarthritis or in normal volunteers. Gelatin
separation led to recovery of approximately 50%
of total lymphocytes. Gelatin separated cells used
in these experiments gave essentially the same responses to the doses of mitogens used on lymphocytes that were further purified by ficoll/tryosil
gradients, indicating that contaminating red cells
and granulocytes in our cultures were not altering
the responses obtained. Time- and dose-response
514
$
4
3
2
1
0
Autologous serum
-
f1
- N
OA N+OA RA
Homologous serum
-
111;1
N OA N+OARA
Fig 1. Showing the mean and standard deviation
of the response to Conconavalin A in the various
patient groups. Normal controls (N), osteoarthrosis (OA), a pool of controls (N 4- OA) and
patients with rheumatoid arthritis (RA). In general, responses are greater when the cultures are
performed in the presence of al;tologous serum,
but the relative differences between RA and controls remains unchanged.
curves were not performed on patient cells so that
different responses in some groups of patients could
reflect differences in the time course of responses
or the sensitivity to mitogens, and not absolute
changes in the proportion of responsive cells.
Statistical analysis of differences between groups
was compared by Student's t test.
RESULTS
Studies in Pooled Homologous Serum
Patients with osteoarthritis did not differ
significantly from normal controls in their
response to Con A, PHA or in mixed lymphocyte culture (MLC) (Table 1). T h e mitogenic response to PWM was lower than
normal, and this difference was significant
at the 0.05 level of probability. Patients
with rheumatoid arthritis showed a striking depression in response to Con A, T h e
mean Con A response was half that of normals, or of the entire control group. Approximately 300/, of the individual observations fell below the lowest values observed in either control group (Figure 1).
?'his depressed response was characteristic
of the population, and was not attributable
to a few very low responders. Figure 2
Arthritis and Rheumatism, Vol. 17, No. 5 (September-October 1974)
UI
9
3
0
2
I
n
a
m
*
3097 f 1898'
44
CON A
6137 f 3448
5308 f 1989
5734 f 2826
17
17
34
No. of Patients
No. of Patients
CON A
17
6937 f 2797
Normal
17
5979 f 1857
Osteoarthrosis
34
6428 f 2356
Combined Normal and
osteoarthrosis
44
3329 f 1806*
Rheumatoid Arthritis
Results expressed as arithmetic mean f SD
*Significantly different from normal, osteo or combined P = < 0.001
tsignificantly different from normal, osteo or combined P = between 0.5-0.01
$Significantly different from normal P < 0.05 but not from each other
Group
PHA
5718 f 2206f
7148 ?z 2483
7673 +- 2468
7409 f 2458
1434f 819$
6316 f 2346t
PWM
1225 f 8391
3037 f 1590
1843 f 1386$
2413 f 1589
*
PWM
2946 f 1562
1819 +. 1248$
2326 f 1567
8762 f 2653
8113 f 2259
8438 2448
PHA
Table 2. Response to Mitogens in Autologous Serum
Results expressed as arithmetic mean SD
*Significantly different from normal, osteo or combined P = < 0.001
?Significantly different from normal, osteo or combined P = between 0.05-0.01
$Significantly different from normal P < 0.05 but not from each other
Normal
Osteoarthrosis
Combined Normal and
osteoarthrosis
Rheumatoid arthritis
Group
Table 1. Response to Mitogens in Homologous Serum
MLC
6061 f 3015
7664 f 2934
6880 f 3522
7180 f 3270
MLC
4459 -C 2971
5050 f 2613
5891 f 2332
5589 f 2540
B
a
i
4
3
a
F
a
:
8
LANCE ET AL
MLC, although lower, were not significantly different from either control group.
> 10
0
*
w
=
znc
LL
-
9-
8 7 -
6 -
5 4 3 2 10- -
01 2 3 4 5 6 7 8 9
Fig 2. A plot of the distribution of responsiveness
to Conconavalin A in the presence of autologous
serum. On the abscissa are counts per minute
X 10'. On the ordinate the number of observations within a given interval are indicated. The
observations are normally distributed.
shows that the frequency distribution curve
had a normal bell shape. Statistical analysis
of this difference revealed a probability of
less than 0.001 when compared to normal
patients, patients with osteoarthrosis, or a
pool of the two control groups.
Rheumatoid patients also had a diminished response to P H A when compared to
both controls. This difference, although
statistically significant (0.05 < P < 0.01),
was far less striking than that observed for
Con A. T h e response to PWM was lower in
patients with rheumatoid arthritis than in
normal patients but was comparable to values found for patients with osteoarthrosis.
Patients with rheumatoid arthritis were
able to respond adequately to histocompatibility antigens in vitro and values for
516
Studies in Autologous Serum
In general, responsiveness to the four
stimulants was better when cells were cultured in autologous serum (Table 2). T h e
greatest relative change occurred in the
MLC response. This augmented response
was significant ( P < 0.05) only for the rheumatoid group. Although the overall trend
was for higher responses, a few patients
with rheumatoid arthritis showed striking
depression of MLC in autochthonous serum. I n at least one case this same effect
was reproduced when cells from normal individuals were cultured in the presence of
this serum. Similar observations have been
reported by other workers (6). There appeared to be a small subgroup (7 patients)
with very low responses (less than 3000
counts per minute) which fell outside of
the normal distribution curve (Figure 3).
These low responders in MLC were not
necessarily low responders to PHA or Con
A, but were found to have very low levels
of total serum complement (7).
OTHER OBSERVATIONS
T h e results tabulated in this report represent only initial studies of each patient.
However, a number of individuals in all
three groups was studied on more than one
occasion. T h e results in these instances
were quite consistent-low
responders remained low, etc.
We have had the opportunity to study
other patients with miscellaneous diseases.
Two comparisons are worth mentioning. A
small group of patients with Crohn's disease (5 cases) showed response pattern similar to that of patients with rheumatoid
arthritis-eg, very low Con A, slightly depressed P H A and normal MLC responses.
Arthritis and Rheumatism, Vol. 17, No. 5 (September-October 1974)
IMMUNOLOGIC REACTIVITY IN RA
R E S U L T S I N MLC
I RHEUMATOID
:oNntas
12
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Con A responses but very depressed PHA
and MLC responses.
The background counts observed for control cultures with cells alone were extremely low compared to stimulated cultures, and
can therefore be ignored. Furthermore,
there were no significant differences in
spontaneous background activity between
the various study groups in either autochthonous or allogeneic serum.
I
m
I
8
I
8
I
m
Fig 3. A comparison of the results in mixed
lymphocyte culture for pool controls (N -k OA)
and patients with RA. Note the 7 very low responses in the RA group.
Patients with lupus erythematosus (6 individuals), on the other hand, had a very different pattern, characterized by normal
DISCUSSION
Peripheral blood lymphocytes from patients with rheumatoid arthritis are extremely hyporesponsive to the mitogen Con
A. A lesser degree of depression occurs with
PHA, but normal reactivity in MLC was
found. These results are compatible with
the possibility that an abnormality of immunologic responsiveness plays some role
in the pathogenesis of rheumatoid arthritis.
Knowledge of the immunologic status of
patients with rheumatoid arthritis is relatively scanty and inconclusive. Some workers have reported depressed cell mediated
immunity, as measured by delayed hypersensitivity to common skin test antigens (811). However, Block et a1 (12) found no differences between patients with rheumatoid
arthritis and hospital controls in reactivity
testing with PPD. Toh et a1 concluded that
the differences they observed could not be
accounted for by age, sex or treatment (6).
Comparing reactivity between different immunopathic conditions showed that depressed responses were most marked for
chronic active hepatitis, less marked in
systemic lupus erythematosus and least affected in rheumatoid arthritis. Denman et
al (13) studied the immunologic performance of two groups of patients with rheumatoid arthritis, comparing these responses
to normal volunteer controls. The patients
with rheumatoid arthritis included those
Arthritis and Rheumatism, Vol. 17, No. 5 (September-October 1974)
51 7
LANCE ET AL
whose disease was severe enough to be
treated with cytotoxic drugs, and a group
of patients with less severe manifestations.
Depressed P H A response (the only mitogen
studied) was evident in patients with severe
disease, but inapparent in the less severe
,group when compared to controls. A similar tendency was found with respect to
evoked antibody response after challenge
with several vaccines. Other studies have
reported normal antibody responses (14) or
a defect in the
M response in patients
with rheumatoid arthritis (15). Quite recently, Rawson and Huang (16,17) have
reported studies similar in many ways to
our own. They found that the in vitro response of peripheral blood lymphocytes,
from patients with rheumatoid arthritis, to
normal allogeneic cells and P W M were
comparable to normal controls, while the
results with P H A were equivocal. Mixed
cultures, using cells from patients with
rheumatoid arthritis as both responders and
stimulators, gave significantly lower responses. They moreover demonstrated normal binding for Con A on the part of rheumatoid lymphocytes, but a reduced capacity
to bind P H A in comparison to normal.
These findings suggest a disturbance in the
representation of lymphocyte subpopulations.
What significance should be attached to
the disproportionate depression of Con A
response noted in this study, and how does
it relate to the evidence cited above of a
functional disturbance of cell mediated immunity?
In mice, the mitogens P H A and Con A
are selective stimulants for cells derived
from the thymus (T) cells (18), and more
recent evidence suggests that they identify
preferentially subpopulations of T cells (19,
20). The precise functional interrelationship between T-cell subpopulations is in518
completely understood, but it seems that
one subpopulation subserves the cell mediated immunities generally attributed to T
cells, while the other may modulate the
magnitude of responses by the former (2124). If the situation in man is analogous to
that in mice, then our findings may identify
a selective lymphoid subpopulation deficiency in the peripheral blood of patients
with rheumatoid arthritis. Recently, Williams et a1 (25) reported a decrease in the
number of cells with the cell surface antigens of T cells in the blood of patients with
rheumatoid arthritis. Such a deficiency could
be relevant to the etiology and pathogenesis
of rheumatoid arthritis in several possible
ways. It has been suggested that autoimmune disease may arise from failure of regulation on the part of T-cell subpopulations (26). The disproportionate depression
of Con A reactively might indicate a deficiency in a T-cell subpopulation, which
should subserve an essential regulatory role
and in the absence of which connective tissue disease subvenes. Alternatively, selective
T-cell deficiency could interfere with the
ability to respond to and eliminate a viral
pathogen, turning a self-limited infection
into a chronic one (27).
T h e defect in responsiveness we have
identified characterizcs the population of
peripheral blood lymphocytes. We do not
know whether this defect represents responses of central lymphoid tissue as well.
Nor do we know whether these changes are
the cause or effect of disease. It is possible
that this response pattern will be found in
many diseases in which chronic granulomata are prominent. The explanation could
be as trivial as sequestration of the relevant
cell population in the granulomatous tissue
producing, therefore, a relative deficiency
in the periphery. The many questions raised
in this study will have to be resolved by
Arthritis and Rheumatism, Vol. 17, No. 5 (September-October 1974)
IMMUNOLOGIC REACTIVITY IN RA
further investigation. Promising approaches
include correlating these findings with other parameters of i m m u n e function, characterizing i m m u n e responses in other granulomatous disease, serial study of early cases
a n d study of parents and siblings of patients with rheumatoid arthritis. A number
of these approaches are already being studied, a n d will be the subject of a further
report.
ACKNOWLEDGMENTS
The authors would like to acknowledge the help
of Drs. M. Gumpel, D. Smith, and B. Porter for
allowing us to study their patients, Dr. J. Abbosh
and Mrs. P. Ridyard for assembling the data, and
Mrs. V. Price for typing the manuscript. Excellent
technical assistance was provided by Miss J. O’Rrien,
Mr. A. Pindar and Miss N. Yianni.
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Arthritis and Rheumatism, Vol. 17, No. 5 (September-October 1974)
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Arthritis and Rheumatism, Vol. 17, No. 5 (September-October 1974)
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