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Study of thymic factors. II. Failure of thymosin to alter the natural history of nzb and nzbnzw mice

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862
STUDY OF THYMIC FACTORS
11. FAILURE OF THYMOSIN TO ALTER THE NATURAL HISTORY OF
NZB AND NZB/NZW MICE
M. ERIC GERSHWIN. ALFRED D. STEINBERG, AFTAB AHMED, and CRAIG DERKAY
The effects of daily injections of thymosin, bovine
fraction V, on the natural history of NZB and
NZB/NZW F, mice were investigated. With the use of
several dose schedules, no significant differences were
discovered in treated versus control groups when survival,
autoantibodies, and mitogen responsiveness were compared. These results provide further evidence that thymosin may have little or no role in the treatment of the
autoimmune disease of New Zealand mice. More encouraging research in thymic extracts and their measurement is necessary before clinical trials in SLE are considered.
In 1959, spontaneous autoantibody production
was found in New Zealand mice (1). This discovery
represented the first naturally occurring animal model of
human autoimmune disease. New Zealand black (NZB)
mice develop Coombs’ positive hemolytic anemia, memFrom the Section of Rheumatology, Department of Internal
Medicine, University of California, Davis, California; the Arthritis
and Rheumatism Branch, National Institute of Arthritis, Metabolism
and Digestive Diseases, National Institutes of Health, Bethesda,
Maryland; and the National Naval Medical Research Institute, Bethesda, Maryland.
M. Eric Gershwin, M.D.: Assistant Professor of Medicine,
University of California, Davis; Alfred D. Steinberg, M.D.: Senior
Investigator, NIAMDD, NIH: Aftab Ahmed, Ph.D.: Chief, Cellular
Immunology, National Naval Medical Research Institute: Craig Derkay: Medical Student, NIAMDD, NIH.
Address reprint requests to M.Eric Gershwin, M.D., Section
of Rheumatology-TB 171. Department of Internal Medicine, University of California, Davis, California 95616.
Submitted for publication November 10, 1975; accepted January 9, 1976.
Arthritis and Rheumatism, Vol. 19, No. 5 (September-October 1976)
branous glomerulonephritis, and lymphoid infiltration
of many organs (2,3). The mating of NZB with NZW
mice produces an F1 hybrid (NZB/NZW) that develops
high titers of antinuclear antibodies and severe immune
complex renal disease (3,4). Most hypotheses attempting to explain these phenomena revolve around the interaction of genetic and viral factors and associated
premature thymic involution (2). In particular there has
been considerable attention to the possibility that a major cause of the dysimmune features in New Zealand
mice is a premature loss of thymic hormone(s) (5-7).
Evidence supporting the concept of loss of thymic hormone(s) in New Zealand mice includes an earlier
report that thymic factor levels are markedly reduced in
NZB and NZB/NZW mice ( 5 ) . Furthermore thymosin,
bovine fraction V, improves some but not all T-helper
cell functions and restores normal thymocyte proliferative and suppressive function (6,7). Similarly,
thymosin has been reported to restore the low level of E
rosettes in humans with SLE to normal levels (8). in
contrast limited trials of thymosin in NZB X NZW mice
indicate no difference in titer of antibodies to nucleic
acids in older animals (9). Finally, the specificity of
thymosin and the significance of the apparent low levels
of thymic factors have recently been questioned (10,ll).
Because thymosin is being considered for clinical trials
in patients with SLE, we initiated a large study to
investigate thoroughly the effects of several dosage
schedules on cellular and serologic abnormalities and
survival in NZB and NZB/NZW mice. We report
863
herein no statistically significant benefit of thymosin on
the natural history of New Zealand mice and caution
against its use in patients with autoimmune disease until
more encouraging research reveals further data.
MATERIALS AND METHODS
Animals. NZB (H-2d) and NZB X NZW (H-ZdIZ)mice
were obtained from the Rodent and Rabbit Production Unit
at the National Institutes of Health, Bethesda, Maryland.
Reagents. Thymosin, bovine fraction V, was the generous contribution of Dr. Allan Goldstein. It was stored lyophilized at -20°C and aliquoted for use in RPMI-1640 media
(Grand Island Biological Company, Grand Island, New
York). The activity of this thymosin preparation was confirmed and periodically checked by means of the modified
mouse rosette assay and the nude mouse lymph node assay as
previously described (10,12). “C-double-stranded DNA was
prepared from KB cells (13). The purity of the native DNA
was tested periodically by reaction with rabbit anti-singlestranded DNA. Such antibody precipitated less than 10% of
“C-KB DNA. Single-stranded DNA was prepared by heating
native DNA for 12 minutes at 100°C and then rapidly plunging, while stirring, into an ice-water mixture. “C-Poly I . Poly
C was purchased from Miles Laboratories, Kankakee, Illinois.
Phytohemagglutinin-P (PHA-P) and Escherichiu coli lipopolysaccharide (LPS) were obtained from Difco Laboratories, Detroit, Michigan. Concanavalin A was purchased
from Calbiochem, San Diego, California, and was dialyzed
against phosphate-buffered saline for 3 days. Urine dipstix
were manufactured by Ames Laboratories, Elkhart, Indiana.
SURVIVAL STUDIES
Groups of 48-100 I-month, 2-month, 3-month, and 6month NZB/NZW female mice were randomized, in four
equal groups, to receive daily injections (7 days/week) of
either a ) RPMI media ip, b) 50 pg thymosin ip, c) 75 pg
thymosin ip bid, or d) 250 pg thymosin ip. All mice were
followed daily for survival.
At ages 5.5 and 7 months the above NZB/NZW mice
were carefully bled by orbital sinus puncture and the sera were
allowed to clot. Antibodies to DNA and Poly I . Poly C were
determined by modification of the Farr technique (14, 15).
Known positive and negative samples were included with each
assay. Comparisons between groups were performed by means
of Students t test.
Proteinuria. At monthly intervals beginning at 5
months of age, all mice were tested for proteinuria following
manual bladder massage. The geometric means between
groups were determined and the results were compared by a
rank sum test and the Students t test.
Mitogens and Mixed Lymphocyte Reactions. At age 7.5
months, 3 NZB/NZW mice treated from age 1 month were
sacrificed by cervical dislocation. Single-cell suspensions of
spleen cells were prepared and cultured in microtiter plates
(1 5). To triplicate cultures were added 100 pI of each mitogen
(phytohemagglutinin-P (PHA-P), 0.1% final concentration,
concanavallin A (Con A), 0.25 pg/culture, E coli lipopolysaccharide (LPS), 50 pg/culture, or media alone (16).
Cultures were incubated for 72 hours at 37°C in a 5%
CO,. 80% oxygen, 15%nitrogen humidified-atmosphere. Eighteen hours before harvest each culture received 1 pCi of
methyL3HTdR, 1.9 Ci/rnMole (Schwarz- Mann, Orangeburg,
New York) in 20-pl media. Cultures were harvested with an
automated multiple sample harvester. Other aliquots of spleen
cells were used, in one-way mixed lymphocyte reaction (MLR)
with 4 X 106 NZB/W responder cells and 4 X 106 rnitomycin
C-treated C3H/HeJ target spleen cells. Cultures were incubated in triplicate and incubated 92 hours before being
pulsed and handled as above.
Effect of Thymosin on NZB,Mice. Groups of 10 NZB
male mice, 1 month and 3 months of age, were randomized to
receive daily injections of a) media; b ) thymosin, 50 pg ip; c)
thymosin. 250 pg ip; or d) thymosin, 250 pg ip every Monday. Mice were bled monthly by orbital sinus puncture for a
microdirect Coombs’ test (17). Sera were examined for presence and titer of antibodies to thymocytes (NTA) (18).
RESULTS
There were no statistically significant differences
in survival of N Z B X N Z W F, mice receiving doses of
thymosin ranging from 50 p g t o 250 p g per day. This
result occurred irrespective of when therapy was instituted. Similarly, the percentages of binding of D N A
and RNA in control and treatment groups were virtually identical (Table 1). Finally, all treatment groups
had the same degree of proteinuria (data not shown).
Spleen cells from 7-month NZB X N Z W mice
have a loss of responsiveness to the T-cell mitogens
Concanavalin A and Phytohemagglutinin-P (Table 2).
They also fail to be stimulated in a mixed lymphocyte
reaction, in contrast t o 1-month animals in whom “normal” stimulation is evident. Thymosin fails to alter both
this age-related hyporesponsiveness a n d the increased
background (autostimulation) (Tables 2 and 3).
N Z B mice treated with thymosin develop circulating, naturally occurring thymocytotoxic antibody in
the same titer as do control mice (Table 4). Similarly, all
mice, irrespective of treatment, develop a positive
Coombs’ test (Table 4). Antibodies t o single-stranded
D N A , for mice 5 months of age, were identical in treatment a n d control groups. T h e fact that the number of
mice in each group was limited (10) precluded statistical
conclusions regarding survival.
DISCUSSION
New Zealand mice represent the first naturally
occurring animal model of human autoimmune disease.
GERSHWIN ET AL
to present with clinically advanced murine lupus (at
about 6-7 months), therapy would be less effective. Thus
for both murine and human lupus more efficacious
means of therapy are required.
Thymic factors have been described for over 30
years (20). However earlier attempts at isolation of a
“hormone” were discouraging because of the crudeness
of the extract and inability to quantitate its function.
Recently, A. Goldstein (21), G. Goldstein (22), Trainin
(23), and Bach (24) have individually prepared considerably purer thymic extracts known respectively as
thymosin, thymopoietin, THF, and thymic factor. None
of these preparations is totally pure. However all share a
number of critical biologic properties, including improvement in T-cell function and restoration of T-cell
markers in thymic-deprived animals (20). Moreover
thymosin therapy of a child with Nezelof syndrome
(lymphopenia with normal immunoglobulins) has been
reported to be partially successful (25).
Methods of measuring thymic factor(s) have
been cumbersome and time-consuming. At present no
reliable radioimmunoassay exists, although an overwhelming effort is being directed toward that purpose.
Bach and coworkers have reported that use of a mouse
rosette assay results in premature loss of thymic factor
levels in New Zealand mice and patients with SLE ( 5 ) .
More recently, the specificity and significance of this
assay has been questioned (10). Thus the basis of thymic
hormone deficiency in murine and human SLE appears
in doubt. Moreover the biologic significance of possible
thymic hormones(s) depletion in NZB mice has also
been questioned (1 1).
In this study no significant improvement in natural history of either NZB or NZB X NZW F, mice
receiving thymosin could be determined. Parameters
measured included antibodies to native DNA and Poly I
Poly C; proteinuria, mitogen, and MLR responsiveness, and survival in NZB X NZW mice; and Coombs’
positivity and production of NTA in NZB mice. It is
conceivable that other dose schedules or different
Table 1. Effects of Thymosin on Natural History of
NZB X N Z W F, Mice
“C Poly
Age of
Mice*
(months) Doset
I
Media
I 5 0 p g q d
I75pgbid
1
250pgqd
Media
2
2
50pgqd
2
75pgbid
“C DNA, % Binding
1 Poly C
%Binding
5.5 Months 7 Months 7 Months
33f6
37f8
32f9
39 f 6
42f3
31 f 8
43 f 7
4 4 f 10
36 f 5
42 f 9
39 f 6
40 f 9
43 f 9
38f6
34 f 9
37%I I
43f7
50f5
54f8
47f9
55f6
5 6 4 10
48 f 9
46f3
47f9
50f7
48f8
52f6
56f9
51 f 7
58f6
57 f 9
63f7
61f9
54fII
6 6 f 13
61 f 14
4 8 f I2
56f II
51 f 14
58f9
50f7
63f9
63f7
5 9 f 18
5 6 f 12
49f I I
53 f 10
Survival$
(mean
days)
325
310
315
330
310
305
335
310
305
315
295
3 10
320
315
320
330
* Ageat onset ofexperiment.
t Thymosin, bovine fraction V.
$ There were 12-25 mice per treatment group. No significant survival
differences were noted with the Kolmogorov-Smirnov test.
The striking similarities between NZB mice and idiopathic acquired hemolytic anemia, and NZB X NZW F,
mice and systemic lupus erythematosus, have been well
reviewed (2). A major advantage of these mice has thus
been that they can be studied in new drug trials before
patients are subjected to clinical usage. As such, therapy
of large numbers of these mice has been performed in
several laboratories and the data have been invaluable
to clinicians (1 9). Although immunosuppressive therapy
of New Zealand mice can markedly retard development
of autoantibody formation and immune complex disease, treatment is more effective when initiated before
clinical disease is manifest-that is, in I-month mice
before detectable anti-DNA antibody is produced.
However, as with patients, if a New Zealand mouse were
Table 2. Incorporation of SH-Thymidineby 7-Month-Old NZBl W Spleen Cells
Treatment*
Media
50 pg/day
75 pg bid
250 pg qd
Control cellst
* Seven-month-old
Background
1,840 f 290
1,775 f 440
2,120 f 310
1,950 f 230
6 4 0 f 170
Con A
PHA-P
LPS
2,370 f 490
2,010 f 380
1,840 f 450
2,210 450
44,300 f 10,300
1,470 f 650
2,3 10 f 550
2,050 f 370
1,810 f 310
85,700 f 14,600
11,300 f 2.500
14,200 f 3,200
12,700 f 1.800
13,600 f 2,100
17,300 f 2,100
*
NZB/W mice treated beginning at age I month with thymosin bovine fraction V.
t Four-week-old NZB/W mice.
THYMOSIN IN NZ MICE
865
Table 3. Mixed Lymphocyte Reaction in Spleen Cells of NZBl W Mice
~
Treatment*
Stimulator
Responder
Uptake of
‘H -Td R ,
(cpm i SE)
C3H
CaH
NZB/W
NZB/W
NZB/W
NZB/W
NZB/W
NZB/W
NZB/W
NZB/W
C3H
1,100 f 143
103 f 39
1,419 f 173
1,317 f 197
1.286 f 139
983 f 246
1,511 f 177
1,283 f 133
1,605 f 243
8,475 f 1, I30
RPMI
RPMI
RPMI
50 &day
75 pg bid
250 rg qd
50 rg/day
75 pg bid
250 fig qd
RPMI
-
C3H
C3H
C3H
NZB/W
* Seven-month-old NZB/W mice treated beginning at age 1 month
both single-stranded DNA and Poly A . Poly U functioned better than thymosin in the mouse rosette assay
(10). The survival data and antibodies to nucleic acids
parallel those in shorter trials reported hitherto (9).
The extrapolation of data from mouse to man
should be done only with extreme caution, Thymosin
remains at present a crude extract and could conceivably
contain components-i.e., minor degrees of nucleic acid
contamination-that might mask any beneficial effects
in long-term therapeutic trials in New Zealand mice.
However, because New Zealand mice and perhaps human lupus patients are immunologically hyperresponsive, new agents must be administered very carefully to
avoid exacerbating their disease.
with thymosin bovine fraction V.
routes of administration could improve these findings.
However doses employed are comparable to the amount
given in vivo to restore MLR responsiveness of congenitally (athymic) nude mice (26). Additionally, intraperitoneal administration of thymosin has shown no
advantage over intravenous administration in a nude
mouse lymph node biologic assay. Earlier data indicated
that short-term treatment of NZB X NZW mice restored the responsiveness of lymph node cells but not of
spleen cells to the T-cell mitogens PHA-P and Con A
and to stimulation in one-way MLR ( 6 ) . It also improved, but did not return to normal, the response to
sheep red cell immunization and skin allograft rejection ( 6 ) . These responses were not examined in this
study in order to reduce the artifacts of manipulation
and to permit survival studies. Indeed these latter events
may be due to a nonspecific adjuvant effect of either
thymosin or nucleic acid contamination. For example,
Table 4. Effect of Thymosin on Natural Hislory of NZB Mice
Age*
I
3
Dose
Positive
Coombs’
Positive
Tests at 5
NTA at 4
Months (%) Months (%)
-
100
100
50 pg qd
250pgqd
250pg q weekly
I00
100
I00
100
100
I00
I00
100
100
-
50 r g qd
250 pg qd
250 pg q weekly
* Age at onset of experiment.
t Measured at 5 months of age.
100
100
100
100
100
SS DNA?
Bound (70)
30 f 8
37 z t 8
40f21
26 f 9
40% 12
3 6 f 11
3 5 f 13
39 f 12
REFERENCES
1. Bielschowsky M, Helyer BJ, Howie JB: Spontaneous
anemia in mice of the NZB/BL strain. Proc Univ Otago
Med Sch 37:9-11, 1959
2. Talal N, Steinberg AD: The pathogenesis of autoimmunity in New Zealand black mice. Curr T o p Microbiol Immunol 64:79-103, 1974
3. Helyer BJ, Howie JB: Renal disease associated with positive lupus erythematosus tests in crossbred strain of mice.
Nature 197:197, 1963
4. Lambert PH, Dixon FJ: Pathogenesis of the glomerulonephritis of NZB/W mice. J. Exp Med 127507-522,1968
5. Bach J F , Dardenne M, Salomom JC: Studies on thymus
products. IV. Absence of serum thymic activity in adult
NZB and (NZB X N Z W ) F, mice. Clin Exp Immunol
10:247-256, 1973
6. Gershwin ME, Ahmed A, Steinberg AD, et al: Correction
of T cell function by thymosin in New Zealand mice. J
Immunol 113:1068-1071, 1974
7. Dauphinee MJ, Talal N, Goldstein Al, et al: Thymosin
corrects the abnormal D N A syhthetic response of NZB
mice. Proc Natl Acad Sci USA 71:2637-2641, 1974
8. Scheinberg MA, Cathcart ES, Goldstein AL: Thymosininduced reduction of “null cells” in peripheral blood lymphocytes of patients with systemic lupus erythematosus.
Lancet I :424-426, 1975
9. Talal N , Dauphinee M, Pillarisetty R, et al: Effect of
thymosin on thymocyte proliferation and autoimmunity
in NZB mice. Ann N Y Acad Sci 249:630-649, 1975
10. Gershwin ME, Steinberg AD, Woody J, et al: Study of
thymic factors. I. Evaluation of the mouse rosette assay. J
Immunol 115:1444, 1975
11. Dauphinee MJ, Palmer DW, Talal N: Evidence for an
abnormal microenvironment in the thymus of New Zealand black mice. J. Immunol l15:1054-1059, 1975
12. Thurman GB, Steinberg AD, Ahmed A, et al: Effect of
thymosin treatment in vivo; increased mitogenic responsi-
GERSHWIN ET AL
13.
14.
15.
16.
17.
18.
vity of murine lymph node cells. Trans Proc 7:299-303,
1975 (suppl)
Pincus P, Schur PH, Rose JA, et al: Measurement of
serum DA-binding activity in systemic lupus erythematosus. N Engl J Med 281:701-707, 1969
Farr RS: A quantitative immunochemical measurement of
the primary interaction between "ll-BSA and antibody. J
Infect Dis 1 05:239-262, 1958
Steinberg AD, Pincus T, Tala1 N: DNA-binding assay for
detection of anti-DNA antibodies in NZB/NZW F1mice.
J Immunol 102:788-791, 1969
Strong DM, Ahmed A, Thurman GB, et al: J Immunol
Meth 21:279-288, 1973
Gershwin ME, Steinberg AD: Suppression of hemolytic
anemia in New Zealand mice. Clin Immunol Immunopatho1 4:38-45, 1975
Shirai T, Mellors RC: Natural thymocytotoxic autoantibody and reactive antigen in New Zealand black and
other mice. Proc Natl Acad Sci 68:1412-1415, 1971
19. Steinberg AD: Efficacy of immunosuppressive drugs in
rheumatic diseases. Arthritis Rheum 18:72-96, 1973
20. Trainin N: Thymic hormones and the immune response.
Physiol Rev 54:272-315, 1974
21. Goldstein AL, Slater FD, White A: Preparation, assay
and partial purification thymosin. Proc Natl Acad Sci
USA 56:1010-1017, 1966
22. Goldstein G : Isolation of bovine thymin: a polypeptide
hormone of the thymus. Nature (Lond) 247:ll-14, 1974
23. Trainin N, Small M: Studies on some physiochemical
properties of a thymus humoral factor conferring immunocompetence of lymphoid cells. J Exp Med 132:885897, 1970
24. Bach JF, Dardenne M: Thymus dependency of rosetteforming cells, evidence for a circulating thymic hormone.
Trans Proc 4:345-350, 1972
25. Wara DW, Goldstein AL, Doyle NE, et al: Thymosin
activity in patients with cellular immunodeficiency. N
Engl J Med 292:70-74, 1975
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