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Lymphocyte response to mitogens in progressive systemic sclerosis.

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875
LYMPHOCYTE RESPONSE TO MTTOGENS
IN PROGRESSIVE SYSTEMIC SCLEROSIS
NOEL B. SALEM and J A N E H. MORSE
Lymphocyte responses to the mitogens phytohemagglutinin P (PHA-P), concanavallin A (Con A), and
pokeweed (PWM) were studied in 18 patients with progressive systemic sclerosis (PSS). A subgroup of these
patients with multisystem involvement showed a significantly decreased lymphocyte response to both Con A and
PWM when compared to normal controls. However those
PSS patients with myositis, although having multisystem
involvement, had normal lymphocyte response to all three
mitogens. PHA-P stimulation was normal in all PSS patients. Antinuclear antibodies, elevated sedimentation rates,
and positive latex fixation were noted only in the multisystem disease group with abnormal lymphocyte function.
Of the four previous studies (1-4) of cellular
immunity in progressive systemic sclerosis (PSS), three
were directed toward the study of lymphocyte stimulation by the nonspecific mitogens. These reports have
noted no abnormalities in cell-mediated immunity
(CMI) with respect to mitogen stimulation. A fourth
study noted increased lymphocyte stimulation by muscle
From the Edward Daniels Faulkner Arthritis Clinic of Columbia Presbyterian Medical Center, New York, New York.
Supported by USPHS Grants HL 10049 and AI-00440 and by
the Lupus Erythematosus Foundation.
Noel B. Salem M.D.: Rheumatology and Immunology Fellow, Columbia Presbyterian Medical Center; Jane H . Morse. M.D.:
Associate Professor of Clinical Medicine. Columbia University College of Physicians and Surgeons.
Address reprint requests to Noel B. Salem, M.D.. Columbia
Presbyterian Medical Center. 630 West 168th Street, New York. New
York 10032.
Submitted for publication December 2, 1975: accepted March
13. 1976.
Arthritis and Rheumatism, Vol. 19, No. 5 (September-October 1976)
extract in patients with PSS (2). Lymphocytes from
these patients were also reported to be cytotoxic to
cultures of fibroblasts and to epithelial and muscle cells.
The current report continues the investigation of
lymphocyte function in patients with PSS to determine
whether the extent of disease involvement is correlated
with altered CMI. The previous studies of lymphocyte
transformation to the nonspecific mitogens (1-4) have
not shown any abnormalities, but they did not group
patients by extent of disease activity. Such grouping has
been shown to be an important variable with respect to
lymphocyte function ( 5 , 6 ) in other autoimmune diseases. i.e., the conflicting data reported on lymphocyte
transformation in SLE and R A (7-12) may be explained
in part by the grouping of patients in this manner.
The findings reported here show that a group of
patients with PSS and multisystem involvement have
decreased lymphocyte transformation to Con A and
PWM, but not to PHA, when compared to normals.
Those patients with a more limited systemic involvement, i.e., skin, esophageal, and Raynaud's phenomenon, had normal lymphocyte transformation to all
three mitogens. Also noted was a subgroup of patients
with myositis w h o had normal lymphocyte transformation to the three mitogens when compared to normals.
MATERIALS AND METHODS
Patients
Eighteen patients with unequivocal PSS from the
patient population of the Edward Daniels Faulkner Arthritis
Clinic of the Columbia Presbyterian Medical Center were
SALEM A N D MORSE
876
studied. Those patients who were uremic were excluded. The
diagnosis was confirmed by at least two members of the Arthritis group. Most were women and their ages ranged from 26
to 66 years. Only 2 were on prednisone and 1 was on indomethacin (Indocin) at the time of the study.
PSS patients considered to have multisystem involvement were those who had at least one of the following
systems involved: pulmonary, pericardial, renal. or muscle.
Pulmonary involvement was documented by chest x-ray
and/or vital capacity: renal involvement by angiography: and
pericarditis by echocardiogram. Myositis was determined by
the clinical presentation (muscle weakness and/or tenderness)
and by elevation of creatinine phosphokinase (CPK). Raynaud’s phenomenon or esophageal dysmotility, as determined
by cine-esophagram, were not considered as criteria for placement into the multisystem disease group. Specific data regarding drug therapy as well as laboratory and clinical parameters
are presented in Tables I . 2, and 3.
A control group consisted of normal volunteers
matched for age and sex. None was receiving antiinflammatory or immunosuppressive medication. Each patient in the
study was assigned a control.
Lymphocyte Cultures
Fifty milliliters of venous blood were collected in a
syringe containing 1 ml of benzyl alcohol heparin (1 U/ml),
and the lymphocytes were separated by the method of Boyum
(13) on Ficoll-Conray gradient. (Ficoll was acquired from
Pharmacia Fine Chemicals, Piscataway, New Jersey, and 60%
Conray was obtained from Mallinckrodt Pharmaceuticals, St.
Louis, Missouri.) The separated lymphocytes were washed
three times with Eagle’s minimal essential medium ( M E M )
(Gibco, Grand Island, New York) containing 50 U/ml penicillin and 50 pg/ml streptomycin and 0.1 mM/ml L-glutamine.
The lymphocytes were then resuspended in this media, now
containing 15% unheated autologous or pooled AB plasma,
and cultured in triplicate in microtiter plates (Lindbro IS FB
96, Lindbro Chemicals Co. New Haven, Connecticut). Each
well contained 0.25 ml of medium and 1 X 105 cells and the
appropriate dose of mitogen as described below. The cultures
were placed at 37 O in a 5% CO, incubator for 4 days. On the
fifth day they were pulsed with 1 pCi per well of SH-thymidine
(6.7 Ci/mM) (New England Nuclear Corp, Boston, Massachusetts) for 8 hours. The microtiter cultures were harvested
and the wells washed extensively with saline onto glass fiber
filters (grade 934 A H ) (Reeve Angel, Clifton, New Jersey),
with a Mash I 1 Harvester (14). The filters were dried and
placed in 12 ml of lnstabray (Yorktown Research, Hackensack, New Jersey) in standard scintillation vials and counted in
a liquid scintillation spectrometer (Beckman Model 3320). The
results were expressed as counts per minute (cpm) of 3Hthymidine per culture, and as the mean of the triplicate cultures.
Dose response curves for each mitogen, Con A, PHAP. and PWM. were previously determined in this laboratory
on normal individuals with unheated pooled or autologous
plasma for the same 5-day culture period. PHA-P (Difco Laboratories, Detroit. Michigan) was made to a stock solution
and used in concentration of 0.001 and 0.005 ml/ml ofculture.
Con A (Calbiochem, San Diego. California) was used a t 1 and
5 pglml of culture and PWM (Gibco) at lo00 and 5000 pg/ml
culture. These dosages were selected as representative of abbreviated dose response curves (low and average doses of
mitogens). Trypan blue viability studies were not routinely
done, but overall spot checks showed 8 2 4 8 % dye exclusion
with the three mitogens.
Statistical analyses of the differences between groups
was compared by the student’s t test and the paired student’s t
test.
Table 1. Clinical and Laboratory Parameters of 8 PSS Patients with Multisystem Disease Without Myositis
Patient
VN
JB
BS
RF
ss
ESR
(normal
Age/Sex
Systems Involved Other
Than Raynaud’s, Skin,
Esophageal
40/F
57/F
59/F
45/F
Pulmonary, renal*
Pericardial, pulmonary
Pulmonary, hypertension
Pulmonary, renal
76
26
I15
50
66/F
40/M
58/F
Pulmonary
JM
FH
SG
59/F
Pulmonary
Pulmonaryt
Pulmonary, hypertension, renal
CPK
(normal
< 30)
ANA
Latex
3
+ speckled
3 + speckled
Weakly pos
I : 5,000 S M
Neg
Weakly pos
3
Weaklypos
Neg
Neg
ND
+ speckled
Neg
89
40
68
4
I2
2
+ homogeneous
Neg
+ homogeneous
I : 16,000
EN A
< 50)
Normal
Normal
Normal
Normal
Weakly pos
Neg
Neg
Neg
Neg
I :500 RN P
Normal
Normal
Normal
I : 1,280
Neg
Normal
Medications
None
None
None
Elavil
Guanethidine
Prednisone,
20 mg
Guanethidine
None
Digoxin
Reserpine
lndocin
Diuril
Guanethidine
N o elevation of creatinine phosphokinase was noted, and only 2 patients were on antiinflammatory medications at the time of the study
* Postmortem exam.
t Open lung biopsy.
LYMPHOCYTE RESPONSE IN PSS
877
Table 2. Clinical and Laboratory Parameters of 5 PSS Patients with Myositis with or Without Other Systemic Involvemeni
Patient
Age/Sex
Systems Involved Other
Than Raynaud’s, Skin,
Esophageal
JW
45/M
CM
57/F
KM
40/M
RG
31/F
Pulmonary, hypertension, myositis
Pulmonary, hypertension, myositis
Pulmonary, pericarditis,
myositis
Myositis
MF
35/F
Myositis
ESR
(normal
< 30)
AN A
Latex
ENA
CPK
(normal
< 50)
I : 16,000
Neg
100
None
Neg
Neg
186
Aldomet
Medications
60
+ homogeneous
2 + homogeneous
25
Neg
N eg
Neg
700
None
32
Neg
N eg
Neg
158
15
Neg
N eg
ND
900
Prednisone,
100 mg qod
None
40
2
Only 1 patient was o n high-dose prednisone at the time of the study.
The sedimentation rate was performed by the Westergren method. Antinuclear antibodies were determined by the
indirect immunofluorescence technique ( I 5 ) with mouse liver
cells as a substrate. Latex fixation was determined with the use
of Bacto Latex particles (0.81 ) (Difco Laboratories). Extractable nuclear antigen was determined by the method previously
reported by Hamburger et a1 (16).
RESULTS
Clinical Findings
Peripheral blood lymphocytes (PBL) from 18
patients fulfilling the criteria for PSS were studied for
transformation to the mitogens PHA-P, Con A, and
P W M . All patients had skin involvement a n d 16 had
Raynaud’s phenomenon a n d esophageal dysmotility.
Thirteen also had multisystem disease independent of
Raynaud’s phenomenon a n d esophageal dysmotility.
These I3 patients with multisystem involvement were
divided into two groups depending o n the presence
(Table 2 ) o r absence (Table 1 ) of myositis. Of these
patients, 9 h a d elevated erythrocyte sedimentation rates
(Westergren method), 7 had positive antinuclear antibodies (ANA). a n d 7 h a d positive latex fixation tests.
Only 2 patients (Table 1 ) had antibodies t o extractable
nuclear antigen (ENA).
Although all of the 5 patients with active myositis
illustrated in Table 2 had significant muscle weakness,
this was the predominant feature in only 2. All 5 had
elevated C P K levels a n d 4 had evidence of myositis
confirmed by E M G . This group was heterogeneous with
respect to their serologic abnormalities. T w o patients
had positive A N A s a n d 1 had a positive latex fixation
test.
T h e 5 patients with only skin involvement, Raynaud’s phenomenon, a n d dysphagia are shown in Table
3. These patients had normal ESR, A N A , and latex
fixation texts.
Lymphocyte Cultures
Significantly diminished response t o t h e mitogens
PWM and Con A, but not t o PHA-P, were noted when
patients were divided into groups according to variations in systemic involvement a n d compared to healthy
individuals matched for age a n d sex. Tables 4, 5. a n d 6
present these statistical data, which were derived with
both the student’s t test and the paired student’s t test.
PBL from 13 patients with multisystem disease
(depicted clinically in Tables 1 and 2) exhibited a diminished response t o C o n A a t the concentration of 5
pg/ml (P < 0.04) a n d to P W M a t the concentration of
1000 pg/ml (P < 0.03) in autologous plasma only, as
shown in Table 4. T h e paired student’s t test showed
significant P values for Con A at 1 pg/ml and for PWM
a t 1000 and 5000 pg/mI. No statistically significant differences were noted with any concentrations of PHA-P
in either AB o r autologous plasma. No statistically significant differences were noted in AB plasma t o Con A.
However P W M at one concentration, 5000 pg/ml, gave
significant P values with the paired t test.
Table 3. Clinical and Laboratory Parameters of 5 Patients with PSS
Including Skin, Raynauds Phenomenon, Esophageal Involvement
Patient Age/Sex
LC
IL
KH
DZ
UB
45/M
58/M
26/F
36/F
54/F
ESR
ANA
Latex
ENA
10
Neg
Neg
Neg
Neg
Neg
Neg
Neg
ND
Neg
Neg
Neg None
N D Guanethidine
Neg Guanethidine
Neg Guanethidine
N D Aldomet
16
2
12
15
Medication
Most patients received therapy for Raynaud’s phenomenon.
SALEM AND MORSE
878
Table 4. Response io Miiogens by 13 PSS Paiienis, with Multisystem Disease, Including 5 Paiienis wiih
Myosiiis, in Autologous and A B Plasma
Mitogen
Concentration
Patient?
Controlst
N o mitogens
Con A 5
ConA I
PWM 5,000
PWM 1,OOO
PHA-P 0.005
PHA-PO.OO1
Autologous Plasma
189 f 88
208 f 125
13,932 f 8,655
22,480 f 10,902
9,760 f 2,057
5,623 f 1,300
22, I42 f 2,680
16,830 f 2,169
24,273 f 3,134
15,119 f 2,347
24,894 f 4,Ol I
27,808 f 2,969
32,039 f 4.53 I
23,877 f 3,979
N o mitogens
Con A 5
ConA I
PWM 5,000
PWM 1,000
PHA-P 0.005
PHA-PO.OO1
AB Plasma
155 f 95
134 f 75
8,667 f 7,967
12,983 f 5,758
5,635 f 1,409
3,604 f 3,510
17,868 f 5,703
12,055 f 9,014
14,372 f 5,830
12,562 f 12,571
19,033 f 8,576
20,357 f 10,363
16,253 f 10,275
18,445 f 10,995
_
_
~
_
_
~
~
~~
_
p by
Student
I Test
p by
Paired
t Test
>0.5
<0.04
>0.05
>o. I
<0.03
>0.06
<O.W
<0.02
<0.02
>0.5
>0.5
>o. I
>o. I
>0.5
>0.1
>O.l
>0.05
>0.5
>0.5
>0.5
>o. I
>O.I
>0.1
>0.1
<0.01
>o. I
>0.5
>0.5
_
* These 13 patients are depicted clinically in Tables 1 and 2. Patient VN in Table I did not have PWM or
PHA-P studies.
t Results are expressed as the arithmetic mean
f SD of counts per minute of aH-thymidine incorporation
per culture.
Table 5 . Response to Miiogens by 8 PPS Palienis* wiih Muliisysiem Disease, Excluding the 5 Paiienis
wiih Myosiiis. in Autologous and A B Plasma
Mitogen
Concentration
N o mitogens
Con A 5
ConA I
PWM 5,000
PWM 1,000
PHA-P 0.005
PHA-P 0.001
Patientt
Controlst
Auiologous
I66 f I I7
10,141 f 5,395
3,846 f 2, I55
13,405 f 4,556
11,055 f 4,910
19,195 f 12,671
17,106 f 10,435
Plasma
198 f 101
24,781 f 13,019
11,161 f 7,614
23,093 f 8,758
23,146 f 7.849
23,060 f 7,797
27,661 f 12,388
P by Student P by Paired
i Test
i Test
>0.5
<0.02
<0.03
<0.03
<O.W
>o. 1
>o. 1
>0.5
<0.05
<0.05
>o. I
<0.005
>0.5
<0.01
<0.03
<0.02
<0.005
<O.M
<O.M
<0.02
>0.5
>0.5
>0.5
>0.5
<OM
<0.02
>0.6
>0.2
A B Plasma
No mitogens
Con A 5
Con A I
PWM 5,000
PWM 1,OOO
PHA-P 0.005
PHA-PO.OO1
130 f 73
4,821 f 4,221
2,154 f 1,985
10,014 f 6,098
6,838 f 4,310
16,742 f 5,664
13,461 f 5,627
I73 f 110
14,251 f 6,210
6,956 f 5.01 1
18,909 f 5,668
15,698 f 5,447
15,271 f 6,419
13,772 f 8, I74
* These 8 patients are depicted clinically in Table I . Patient VN in Table 1 did not have PWM o r PHA-P
studies.
t Results are expressed as the arithmetic mean f SD of counts per minute of 3H-thymidine incorporation
per culture.
8 79
LYMPHOCYTE RESPONSE IN PSS
When the lymphocyte cultures of 8 patients with
multisystem involvement excluding myositis (depicted
clinically in Table 2) were analyzed, there was a statistically significant decrease in 3H-thyrnidine incorporation
at both concentrations of Con A and PWM in both
autologous and AB media (Table 5 ) . The data were
statistically significant by both the paired and unpaired
student’s t tests.
Figure 1 illustrates the data of these same 13
patients with multisystem disease in autologous plasma.
This illustration shows the distribution of these patients,
including the 5 with myositis. The lymphocyte response
of the patients with myositis is not significantly different
from that of normals, as shown in Figure 1 and Table 6 .
Table 7 illustrates SH-thymidine incorporation
by lymphocytes from those patients with only skin involvement (depicted clinically in Table 3). In this group
of patients there was no statistically significant difference in lymphocyte response to the three mitogens at
any tested concentration, in either A B or autologous
plasma.
DISCUSSION
The findings in this study indicate that a subset of
patients with PSS have impaired cell-mediated im-
munity (CM I ) as evidenced by decreased lymphocyte stimulation to certain concentrations of PWM and Con A,
but not to PHA. Clinically, this group appears to be
distinguished by multisystem disease and the presence of
serologic abnormalities (positive latex fixation, positive
ANAs, and elevated ESRs). Further subdivision of
these patients into those with myositis and those without
demonstrated that the myositis patients had normal
lymphocyte transformation, despite wide variability in
serologic abnormalities. Therefore the remaining group
with multisystem disease without myositis had even
greater impairment of C M I when compared to normals.
Although these myositis patients clinically overlap with a group of ENA positive patients described as
having mixed connective tissue disease by Sharp et af
(l7,18), no patient in the present group had a positive
ENA. Only 2 of the PSS population studied had positive
ENAs, and they were among the 8 patients with multisystem disease without myositis.
Winkelmann (19) classified patients with PSS
into two categories, cutaneous and systemic. Multisystem disease and serologic abnormalities were associated with a more severe illness. The clinical data presented here are in keeping with this classification. This
does not imply that PSS in the cutaneous subgroup
Table 6 . Response to Mitogens by 5 PSS Patients* with Myositis with or Without Other Systemic
Involvement in Autologous and A B Plasma
Mitogen
Concentration
Controlst
Patient?
N o mitogens
Con A 5
Con A I
PWM 5,000
PWM 1.000
PHA-P 0.005
PH A-P 0.00 I
Autologous
275 f 116
19,999 f 9,952
8,466 f 6,020
2 1,625 f 7.867
20,808 f 7.8 17
32,872 f 10,464
33,357 f 11,087
N o mitogens
Con A 5
ConA I
PWM 5,000
PWM 1,000
PHA-P0.005
PHA-P0.001
I39 f 85
14,820 f 9.07 1
5,925 f 4.372
14.91 I f 12.253
20,574 f 16,405
22,240 f 1 1,492
20,161 f 14,495
Plasma
I70 f 62
18,799 f 5,694
6,959 f 4,429
20,239 f 10. I77
26,528 f 15,562
35,405 f 9,686
40,769 f 17,790
P by Student P by Paired
t Test
t Test
>O.l
>0.8
>0.6
>0.8
>0.4
>0.7
>0.4
>O.l
>0.5
>0.5
>0.6
>0.4
>0.2
>0.9
>0.3
>0.2
>0.4
>0.5
>0.5
>o. I
>0.5
>0.5
A B Plasma
117f39
10,447 f 4,326
2,993 f 2,750
15,786 f 5.969
1 1,720 f 6.429
30,528 f 9,524
27,791 f 10,613
>0.5
>0.5
>o. I
>0.5
>o. I
>0.5
* These 5 patients are depicted clinically in Table 2.
t Results are expressed as the arithmetic mean f SD of counts per minute ofSH-thymidine incorporation
per culture.
880
SALEM AND MORSE
remains limited; such individuals may later exhibit multisystem manifestations. It was in this multisystem disease group that impaired CMI was noted as well as a
more progressive clinical course. Three of those patients
have died, 2 of renal disease and 1 of unexplained coma
with sclerodermatous renal disease noted at postmortem. I n contrast, those patients with cutaneous involvement lacked serologic abnormalities, and in addition this study has shown this group to have normal
CMI. The short-term nature of this study does not allow
prediction of the evolution of serologic abnormalities
and aberrant CMI in relationship to disease activity.
This report agrees with previous studies that
noted normal lymphocyte transformation to PHA (1-4).
However it disagrees with a recent report by Lockshin et
a1 (3) regarding lymphocyte stimulation in patients with
rheumatic diseases. That study included 5 patients with
PSS whose lymphocyte blastogenesis to the three mitogens showed no significant differences when compared
to chronically ill controls. Several reasons may account
for these differences: a ) the small number of patients in
Lockshin’s series: b) the lack of grouping by disease
activity: and c) the use of a chronically ill control group.
Reports of CMI in other connective tissue disease en-
60,000
I
-5zz 50,000
0
2
LL
40,000
2
z 30,000’
W
0
3 20,000
5
2
I-
10,000
-
I
I
m
-
1
P.SS.Cont.
CONA5
tities (5,6) appear to depend heavily upon assessment of
disease activity. The selection of chronically ill patients
rather than healthy individuals might be the more correct control. However, in this study, when the chronically ill patients with multisystem disease and active
myositis were compared to the healthy control group,
they had normal CMI. Therefore this group may be a
more appropriate “chronically ill” control group for
this study.
This study also noted the depressed lymphocyte
response to PWM and Con A by the PBL of patients
with PSS and multisystem disease with the preservation
of a normal PHA response. Lance et a1 have reported a
similar differential response to Con A versus PHA in
patients with rheumatoid arthritis ( 1 2). These findings
may indicate variations in subpopulations of lymphocytes (20.21). Enumeration of T and B lymphocytes
in PSS has shown significant depression of T cells as
measured by sheep red blood cell rosettes in these
patients (4). The differential mitogenic response reported in the current study may also reflect the same
decrease in T cells or a subpopulation. Alternatively,
PHA may represent a qualitatively differing transforming stimulus.
j
.a
xa
-
a’
c
T
.
*
*
L
-
P.S.S.Cont.
P.S.S. Cont.
P.S.S. Cont.
P.S.S.Cont.
P.S.S. Cont.
COMA I
P W M 5 0 0 0 PWM 1000
PHI-P.005
PHA-P.OO1
Fig 1. Comparison of SH-thymidine incorporation by peripheral blood lymphocytes from I3 PSS patients
with multisystem disease to the mitogens PHA-P. P W M , and Con A in autologous plasma. The 8
patients without myositis (Table I ) are depicted by the closed circles and the 5 patients with myositis (Table
2 ) by the crosses. The thin bar is the arithmetic mean of the 8 patients without myositis, who are also
statistically illustrated in Table 5. The heavy bar is the arithmetic mean of the 13 patients who are
statistically illustrated in Table 4. For the control groups, the thin bar represents the arithmetic mean ofthe
corresponding 8 age-matched controls for the nonmyositic patients. The heavy bar represents the arithmetic
mean of the corresponding 13 age-matched controls for the entire group of PSS patients with multisystem
disease.
LYMPHOCYTE RESPONSE IN PSS
88 1
Table 7 . Response to Mitogens by 5 PSS Patients* with Skin, Raynauds Phenomenon. and Esophageal
Involvement in Autologous and A B Plasma
Mi togen
Concentration
Patientt
Controlst
N o mitogens
Con A 5
ConA I
PWM 5.000
PWM 1.000
PHA-P 0.005
PHA-P 0.001
Autologous Plasma
202 f 55
231 f 113
25,007 f 1 1,537
24.787 f 13,721
I 1,464 f 3,605
12,217 f 3.967
26,566 f 9,463
17,509 f 7,205
20,946 f 7,484
28,289 f 14,578
26.559 f 17,594
24,188 f 5,608
22,528 f 6,570
24,660 f 1 1,788
N o mitogens
Con A 5
Con A 1
PWM 5,000
PWM 1,000
PHA-P 0.005
PHA-P 0.001
144 f 71
12,769 f 6,278
4,255 f 2, I 12
13,523 f 6,575
10,468 f 3,858
22.096 f I 1.277
29,113 f 18,417
A B Plasma
I49 f 107
16,344 f 5.903
6,899 f 4.1 19
21,630 f 5,183
18,883 f 4,804
25, I44 f 13,267
19.31 3 f 6,696
P by Student's
t Test $
>0.5
> 0.5
> 0.5
> 0.1
> 0.1
> 0.5
> 0.5
> 0.5
> 0.1
> 0.1
> 0.5
> 0.2
> 0.5
> 0.1
* These 5 patients a r e depicted clinically in Table 3.
t Results are expressed as the arithmetic mean f SD of counts per minute of 3H-thymidine incorporation
per culture.
$ P by paired
t
test
> 0.1 for all determinations.
Studies in SLE and RA have indicated that humoral factors play a major role in the in vitro measurement of CMI (22-24). Specifically antileukocyte antibodies have bcen noted in SLE and R A (22). The studies
presented here, which use an abbreviated dose response
curve in both autologous and A B media, indicate
the existence of cellular defects, because depressed lymphocyte responses were demonstrated in AB as well as autologous media in the
group of patients with multisystem disease without myositis; and
the existence of humoral factors, because lymphocyte stimulation was depressed in patients
with multisystem disease with myositis in autologous media only.
Admittedly, the method of short-term cell culture without subsequent cell washing or medium changes did not
exclude the possibility that serum factors may have been
bound to the surface of the lymphocyte; thus the ability
of the lymphocyte to respond normally may have been
obscured. More definitive studies would result from examination of the effect of serum from patients with PSS
on stimulated normal lymphocytes. Also, because the
lymphocyte cultures were not purified further to elimi-
nate monocytes and mononuclear cells, the defects in
CMI noted here could be attributed to the monocyte
rather than to the lymphocyte.
Interestingly, even though the patients with myositis had multisystem disease, they had normal CMI as
measured in this study. Further experiments would have
to determine whether this group is heterogeneous or
whether suppressive or protective factors might account
for these findings. Conversely, the presence of myositis
may require normal lymphocyte transforming capacity,
in that only those subjects with normal CMI are capable
of developing myositis.
The apparent overlapping clinical picture of PSS
when multisystem involvement is present with the other
autoimmune disease, in which antileukocyte antibodies
have been demonstrated, suggests that these same antibodies could exist in PSS and, if present, could account
for the depressed CMI noted in vitro in this study.
ACKNOWLEDGMENTS
The authors thank Dr. Peter Barland for performing
the ENA analyses. The skilled technical assistance of Georgianne Ochab is gratefully acknowledged, as is the secretarial
assistance of Doris J. Allen. w h o prepared the manuscript.
SALEM AND MORSE
882
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