- Email: [email protected]
Anti DNA antibody production by lymphoid cells of NZBW mice and human systemic lupus erythematosus (SLE)
(‘I.ISICAL
IhNUNOLOGY
AND
I~~~~L’SOPATHOLO(;~
1. 293-303
(1973)
Anti DNA Antibody Production by Lymphoid Cells of NZB/w Mice and Human Systemic lupus Erythematosus (SLE)’
Single-stranded DNA (S-DNA) coupled to sheep erythrocytes by the chromic chloride method was used to detect antibody-producing cells by a standard hemolytic plaque assay (PFC), and antigen sensitive rosette-forming cells (RFC) by the technique of immunocytoadherence. The spleens and other lymphoid tissue oft?l9-month-old NZB/W F, female mice were studied. The spleens of these mice showed anti-S-DNA antibody-producing cells, which reached a peak in the 7-9month-old mice, and correlated directly with the magnitude of the serum antis-DNA antibody detected by the Farr assay. Shrdies of human lymphoid cells from bone marrow and peripheral blood of patients with systemic lupus crythematosus (SI,E) also revealed significant numbers of anti-S-DNA PFC’s.
There is growing interest in the possibility that some autoimmune diseases may develop among those individuals who have an underlying genetic or acquired abnormality in lymphoid cell function (2-4). The nature of this underlying disorder(s) has not been established, although a number of different observations have suggested that an abnormality of thymus or thymus-derived cells may be involved (l-5). Systemic lupus erythematosus (SLE) represents a prototype autoimmune disorder in which a suitable animal model, the NZB mouse, is available for study. The development of techniques useful for study of the spontaneous immune responses to self antigens in these mice should also be useful in the study of such immune responses in humans with SLE. We have developed a reproducible system for studying those aspects of lymphoid cell function represented by plaque-forming cells and rosetteforming cells to single-stranded deoxyribonucleic acid (S-DNA). These techniques offer the opportunity of quantitating numbers of both antibodyproducing cells (PFC), and antigen sensitive cells (RFC) reactive to a welldefined antigen. The response to S-DNA was chosen since (1) this antigen is chemically well defined and readily available in highly purified form, (2) antibodies to S-DNA are present in a large proportion of both NZB/W mice (6,7) ’ This study was supported by NIH Grant AM 1511802. * Medical Research Council of Canada, Fellow, Division Research Foundation. Address after July 1, 1972: Department Ontario, London, Ontario, Canada. n Present address: V. A. Hospital, Minneapolis, Minnrsota. 293 Copyright All rights
@ 1973 hy Academic Press, Inc. of reproduction in any form reserved.
of Rheumatology, of Medicine,
Scripps University
Clinic and of Western
294
HELL
ET
.4L.
and patients with SLE (8-11,22), (3) a number of studies indicated that this is an important potential immunogen in man (12,13), and (4) antibody-antigen complexes involving DNA have been incriminated in the pathogenesis of both NZB/W mice (6,14) and humans with SLE (8,15). Although a number of studies in both humans and mice with SLE have examined serum or tissue fixed anti DNA antibody, no direct studies of the cellular mechanisms supporting the antibody response to this antigen have been reported. Such an approach should allow a direct study of factors effecting production of antibody to DNA both in z:iuo and in Gitro in these autoimmune disorders. In this report we show that S-DNA coupled to sheep erythrocytes (SRBC) may be used in a standard PFC assay to quantitate anti S-DNA antibodysecreting cells in the spleens of NZB/W mice. The same indicator SRBC’s can be used to detect a larger population of spleen cells in these mice which specifically bind to S-DNA, but do not secrete detectable quantities of complement-fixing antibody. Preliminary studies of these data have been presented previously (16). These techniques have been applied to detect antibody-secreting cells in humans with SLE, and a preliminary report of these data are presented here. MATERIALS
AND
METHODS
Animals. NZB/W F, female mice were provided by Dr. Frank Dixon, Scripps Clinic and Research Foundation. These mice were obtained from mating NZB/Bl females with NZW/Lac males, and were obtained from the colony of mice that have been used in previous studies from this institution (7, 14,17). Normal BDF, male mice (C57 B1/6 female X DBA/2 male) were obtained commercially (Jackson Laboratories, Bar Harbor, ME). Plaque-forming cell JPFC) cissay. Plaque-forming cells from spleens or other lymphoid cells of mice or humans were developed by a modification of the standard Jeme plaque assay in gel (18). Normal or DNA coupled SRBC (Colorado Serum Company) were mixed with single cell suspensions of leukocytes in melted 0.4% agarose, then poured onto microscope slides. The slides were incubated in a moist chamber for 60 min at 37“C, then exposed to 10% guinea pig complement (absorbed with SRBC) with or without facilitating serum (see later). The slides were then incubated again for 60 mm at 37°C then at 4°C overnight and read with the aid of a hand lens. In experiments designed to detect 7S PFC, goat anti-mouse 7SyG serum absorbed with SRBC was used. Two different sera were used. One serum was obtained commerically, and a second was a gift from Dr. Bruce Clinton, Scripps Clinic and Research Foundation. These sera were equally capable of maximally facilitating the development of 7S anti SRBC PFC in immunized normal (BDF,) mice. In studies designed to detect 7S PFC of human lymphoid origin, rabbit anti human 7SrG serum absorbed with SRBC was used, This serum was obtained by hyperimmunizing rabbits with human Cohn Fraction II in cornplete Freund’s adjuvant. Rosette-forming ceE1(RX) c~ssay. Rosette-forming cells were developed by
ANTI
DNA
ANTIBODY-PRODUCING
CELLS
295
a modification of the centrifugation suspension technique of McConnel et ul. (19). For these studies the normal or DNA coupled SRBC used for plaquing were mixed in a 20: 1 ratio with washed mouse leukocytes (5-10 x lo6 nucleated cells/ml). This mixture was centrifuged at 1OOg for 15 min at 4°C incubated a further 30 min at 4”C, then gently resuspended in Balanced salt solution (BSS) (20) to a convenient concentration for counting; 3000-5000 nucleated cells were scanned for leukocytes with at least four adherent erythrocytes. Duplicate counts were made in each instance and the average number of RFC/106 nucleated cells calculated. In some instances, the rosettes were stained with Giemsa on microscope slides in order to study the morphologic appearance of rosetting cells. Coupling of DNA to sheep erythrocytes. Calf thymus DNA (Worthington Biochemical Corp., Freehold, N. J.) was rendered single stranded by heating to 100°C for 12 min, followed by rapid cooling in an ice bath. One milliliter of this single-stranded DNA at a concentration of 500 pglml was then mixed with 0.1 ml of 2% chromic chloride (J. T. Baker Chem. Co., Phillipsburg, N. J.), and incubated at ambient temperature with 0.2 ml of 50% washed sheep erythrocytes. The coupled sheep erythrocytes were then thoroughly washed in phosphate buffered saline (PBS) pH 7.3 and prepared as a 7% suspension in BSS for plaquing or stored at 4°C until required. S-DNA was detected on these cells by passive hemagglutination or passive immune hemolysis with rabbit anti S-DNA serum absorbed with SRBC. Derivatization was found to be constant by these means among different cell preparations, and such cells remained stable at 4°C for at least 6 days. Serum anti S-DNA antibody. The serum of both NZB/W and BDF, mice were examined for the presence of anti S-DNA antibody by the sensitive Farr assay. S-DNA used in these studies was sonicated prior to labeling. Sonication was performed with a Branson Sonifier (model S-125 Branson Instruments Inc., Danbury, Conn.) 20,000 kHz at 2.5 A for 2 min. Two micrograms of sonicated single-stranded calf thymus DNA labeled passively with tritiated actinomycin D (4 &i of tritiated actinomycin D per mg of DNA) as previously described, (21,22), was mixed with a 1: 10 dilution of heat inactived test serum for 60 min at 37°C then at 4°C for 18 hr. The mixture was then precipitated by the addition of saturated (NH,),SO, to a final concentration (vol : vol) of 50%, and the precipitate washed with 50% saturated (NH&SO,. The average number of counts per minute of the labeled S-DNA precipitated by each serum was calculated and expressed as the percentage of antigen binding capacity of the serum (ABC). RESULTS The numbers of PFC and RFC in normal BDF, mice are summarized in Table 1. Few anti S-DNA PFC or RFC were detected in these normal mice. Similar studies of PFC and RFC in NZB/W mice demonstrate that a significantly higher number of anti S-DYA reactive cells exist in the spleens of these mice (Table 2). The NZB/W rn; :e showed a range of from 16 to 452 direct (19s) PFC and from 13,000 to 85,000 RFC/106 nucleated spleen cells, the
296
BELL
ET
AL.
TABLE
1
ANTI S-DNA PFC AND RFC IN SPLEENS OF NORMAL (BDF,) MICE” Direct
RFC/lO”
PFC/lO’
“IO(l)” 2 (1)
Av
“2 7.7 7 4.4 7.9 7
4 (1) ‘6.2 (1.5) ‘1 (1) “1 (1) l(l) 1 (1) d3.2 (1.0)
(1.5) (7.7) (7) (9.6) (7.9) (7)
lF(‘5.6, “6.9 (6)
a Data represent PFC or RFC values in pools of two mice per pool Ir Numbers in parentheses indicate background PFC or RFC values values. c Data represent PFC or RFC from single spleens. d Arithmetic mean. e Not tested.
TABLE
except where indicated vs normal sheep RBC
2
ANTI S-DNA PFC AND RFC IN SPLEENS OF UNIMMUNIZED B/W MICE
Age ho) 6 6 6 6 7 7 8 9 9 9 9 9 9 10 10 10 10
PFC/lO” Direct
Anti Indirect
69 (l)*
20 (1) 21 (0) 22 (0) 259 452 253 109 132 214
(1) (4) (2) (1) (4) (2) 16 (1) 20 (1) 168 (2)
83 (59 74 0) 34 (10) 94 (10)
q % ABC is % of labeled the presence of 50% (NH,), b Numbers in parentheses e Not tested.
RFC/
Spleen
% ,4BC’
lo3
wt
(mgl -’
0 0 0 0
-
-( -
24 85 53 28 25 33 36
0 11 18
-
S-DNA SO,. denote
Serum S-DNA
-
(11) (10)
-.
(7) (7) (5) (14)
553
(8) -
-_ 239 114 301 963
82 (88) -
13 (6.3) 55 (3.8) precipitated
by a 1:lO
background
PFC
dilution
or RFC
of heat
vs normal
inactivated
SRBC.
serum
in
ANTI
DNA
ANTIBODY-PRODUCING
297
CELLS
latter representing 6,700 to 75,000 anti S-DNA RFC. The number of anti S-DNA PFC correlated directly with the amount of serum anti S-DNA detected in the Farr assay. The quantity of antibody to S-DNA detected by both of these procedures appeared to be higher among animals 7-9 months of age, although considerable variability existed even among animals of the same age group. None of the animals studied were moribund at the time they were sacrificed. These mice also showed a wide variability in the number of anti S-DNA RFC and spleen weights, neither of which correlated with age or S-DNA antibody production. Attempts to demonstrate 7s anti S-DNA PFC by the addition of varying dilutions of facilitating serum usually resulted in either no facilitation or actual suppression of tota PFC. Only two of the mice studied had detectable 7s PFC demonstrated by this technique. The sera used for these studies were readily capable of facilitating anti SRBC PFC in BDF, mice primed with SRBC. Table 4 summarizes the result of a number of experiments designed to show specificity for S-DNA in the rosette-forming cell assay system. In these experiments spleen cells of individual NZB/W mice were preincubated for 60 min at 4°C with varying concentrations of S-DNA, native DNA, or human serum albumin. These spleen cells were then mixed with normal or S-DNA coupled SRBC in the usual way. Inhibition of anti S-DNA RFC with S-DNA proved more difficult than inhbition of anti S-DNA PFC (Table 3). At the concen-
INHIBITION PFC S-DNA Expt’ 1
2
Av inhib (%) S-DNA PFC Inhib (%) SRBC PFC*
TABLE 3 OF ANTI S-DNA WITH S-DNA Inhibitor
(pg)
Nil
.OOl
.Ol
1
245 119 136 112 38 121 140 141 82
112 71 89 99 -
40 31 8 15 -
-c
-
-
-
4 23 16 12 37
0 7 8 7 27
-
35
82
82
94
-
-
0
6%
-e
n Data represent PFC/lOG nucleated cells in spleens of eight individual separate experiments, and are the average of replicate determinations. * Data represent av % inhibition of direct anti SRBC PFC in three BDF, previously with 0.2 ml 10% SRBC iv. c Not tested.
5
B/W mice
mice
in two
primed
4 days
BELL
298
SPLEEN
ET
AL.
TABLE 4 INHIBITION OF ANTI S-DNA RFC IN NZB/W CELLS WITH S-DNA, N-DNA, AND HUMAN SERUM
ALLWMIX~
Inhibitor
S-DNA (/.d Nil
.Ol
6 8.3 6.5 8.3 27 36 28 30
28 -
34 55
34 -
82
75
Av. 9% Inhib
.05
1
1.3 2.3 -
3
3.3 3.1 2 6 12 10 10 20 24
80
69
N DNA 5
10
2.1 1.3 -
-
-
75
(wg)
.I
HSA 1
5 7
100
5 6 6.8 6.2
1 6 12 3 4 2 9
-. -
-
85
17
17
!cLg)
16 8 -. .-
2
u Data represent net number of RFC/10” vs S-DNA after preincubation of spleen cells from individual mice with denatured DNA (S-DNA), native DNA (NDNA) or human serum albumin (HSA). Numbers represent the average of duplicate determinations.
trations tested, S-DNA showed significantly more inhibition (69-85s) of anti S-DNA RFC than either “native” DNA (17%) or human serum albumin. Distribution of PFC and RFC in lymphoid cells of NZBIW mice. Table 5 shows preliminary studies comparing the distribution of PFC and RFC values in pooled peripheral lymph nodes, spleen, and thymus of two unimmunized NZB/W mice (age 6 mo) compared with three BDF, mice primed 5 days previously with 2 x lo* SRBC intravenously. Although the anti S-DNA response of these B/W mice was relativeIy low, it is clear that spleen cells
DISTRIBUTION NZB/W
NZB/W NZB/W BDF,
TABLE 5 OF PFC AND RFC MICE AND IMMUNIZED
Spleen
PFC/lOG Nodes
21” 7 1610*
1.5 1 2.5
IX UNIMMUNIZED BDF, Mice
Thymus
Spleen
1.5 0 4.5
6.4 6.4 39.
RFC/l@’ Nodes
Thi
0 .2 1.6
.6 .5 1.2 _-
u Net number of anti S-DNA PFC/lOe or RFC/103 nucleated cells. b Direct anti-SRBC PFC/lOB or RFC/lO” nucleated cells in BDF, with SRBC (pooled tissues of three mice).
mice
5 days
after
priming
ANTI
DNA
ANTIBODY-PRODUCING
TABLE
299
CELLS
6
ANTI S-DNA PFC IN HUMAN BONE MARROW AND PERIPHERAL BLOOD LEUKOCYTES PFC/lOs Bone Pt.
Diagnosis
T.R.
SLE
L.C.
SLE
L.F. H.R. D.S. E.B.
SLE SLE SLE SLE Normald
Prednisone Nil 60mg
marrowa~*
Direct
Indirect
4 (0) 251( 105) 2 (0) 8 (3) 0 -
Peripheral Direct
> 1000
(0)
Indirect
0 0 -
5 (0) 0 108 (0) 0 0 0 0
200-400 0 0 0 10 (0) 0 0
-
0
0
0 200-400
(0)
blood* Disease (0)
activity -
++ i +++ 2 ++ * -
a From iliac crest. * Erythrocytes removed by NH&l lysis and cells washed three times in balanced salt solution. c Background PFC vs normal SRBC values are in brackets. d Peripheral blood leukocytes from three normal donors studied as controls in three different experiments with SLE leukocytes.
make the largest contribution to the total number of direct PFC and RFC in both the NZB/W mice and the SRBC primed BDFl mice. Anti S-DNA PFC in human lymphoid cells. Table 6 outlines our experience in employing the foregoing techniques to study antibody-secreting cells in human SLE. In these studies leukocytes from bone marrow and/or peripheral blood of patients with SLE were plated in the usual way with normal or S-DNA coupled sheep erythrocytes and the number of PFC per lo6 nucleated cells calculated. As shown in this table, the bone marrow cells of four of five patients had detectable numbers of PFC above background and in three of these patients large numbers of anti S-DNA PFC were seen. Two of these patients had, in addition, significant numbers of S-DNA PFC in their peripheral blood, These peripheral blood PFC were not detectable in the three normal donors or in the SLE patients after initiation of high dose corticosteroid therapy. The table indicates that both 19s and 7s PFC were identified in these patients, which contrasts with our observations showing a paucity of 7s anti S-DNA PFC in the NZB/W mice. The finding of significant numbers of 7s PFC among some of these human lymphoid cells, indicates that our inability to detect such 7s PFC in the NZB/W mice is probably not related to inadequate derivatization of SRBC with S-DNA. DISCUSSION The data reported here show that sheep erythrocytes derivatized with S-DNA by the chromic chloride method provide convenient target cells for
300
BELL
ET
AL.
studying specific antibody-producing cells and antigen sensitive cells in both humans and NZB mice with systemic lupus erythematosus. No similar studies of S-DNA antibody-producing cells have previously been documented in either mice or humans with SLE. In addition to the obvious interest in the distribution of antibody-forming cells among various compartments of the lymphoid system, there are a number of advantages to studying antibody production at the cellular level. The PFC technique avoids the difficulty presented by antibody being masked by complexing in the circulation with antigen, a phenomenon presumed by others (26,33) to have made it impossible for them to demonstrate antibody and antigen simultaneously in the circulation of some SLE patients. The release of DNA into the circulation following a variety of stresses and corticosteroid therapy and consequent sudden fall in detectable serum anti DNA antibody in patients with SLE (34), may be a further example of this phenomenon. These factors place a limitation on the interpretation of changes in serum anti DNA antibody levels during the course of this disease. The PFC technique on the other hand, allows day by day evaluation of antibody production, which may vary with disease or as a part of the therapeutic regimen. The technique may also theoretically allow one to obtain information on the affinity of antibody molecules being secreted at any given time. Although affinity measurements may be obtained through an examination of serum antibody itself, the masking factors already mentioned may not allow study of those antibody molecules of highest affinity. Such information may be of value in attempting to study the effects of agents designed to manipulate the immune response. The use of PFC and RFC detection should provide a means for assessing tolerance to nucleic acid antigens in experimental animals, and can be expected to provide interesting data on the effects of virus infection on antibody responses. A number of recent studies by Oldstone and collaborators (7,17) have shown that some viruses may accelerate and others depress certain features of the disease in NZB/W mice, including the level of serum anti S-DNA antibody. The mechanism leading to these effects could be profitably studied at the cellular level, in ciao or in citro, since it is presently not known whether viruses alter the form and/or immunologically effective concentration of host nucleic acid or other antigens, or whether they exert their effects by modifying the activity of cells destined to, or already in the process of secreting antibody. These studies reported here, show that anti-SDNA PFC in the spleens of these NZB/W mice peak at 7-9 months of age, a time which corresponds to the period of greatest mortality from glomerulonephritis, reported in these mice (35). Those mice which had survived beyond this period had relatively fewer anti-SDNA PFC. A surprising aspect of the immune response observed in these chronically ill mice is the relative paucity of 7S anti-SDNA PFC. Since others have reported that the predominant class of antibodies detectable in the renal glomerulus of NZB/W mice are in the IgG class, this observation of a predominance of 19s antibody producing spleen cells will require further
ANTI
DNA
ANTIBODY-PRODUCING
500400300cp 0 r. z z .-P i
. .
.
200-
301
CELLS
.
0.
.
.
loo-
. .
40-
.
% Normal
r;,
[BDFl)
NZB/WFl 20-
?*
Miceo Mice*
l
B I 10 20 30 Serum % S.DNA Binding FK.
40 Capacity
50 [ABC]
I 60
1.
study. Our ability to detect 7s anti-SDNA PFC in human lymphoid cells using the same derivatized SRBC, however, make it seem unlikely that this observation is merely related to technical factors in our assay system. While the studies reported here demonstrate a general direct correlation between serum antibody levels, and splenic PFC, no such correlation was seen with the larger population of splenic RFC and either serum antibody or PFC. This observation is consistent with the notion that the anti-SDNA rosetting system used here may be measuring a heterogeneous population of antigen reactive cells. Studies of the exact nature of this population of SNDA reactive cells are needed, with particular regard to their possible origin from bone marrow and/or thymus. The data on antibody-secreting cells in the peripheral blood and bone marrow of our SLE patients are somewhat preliminary, since only a few patients have been studied. Surprisingly no published studies have been made of the antibody producing potential of human bone marrow or human peripheral blood leukocytes using the techniques reported here. Abdou and Abdou have recently presented evidence supporting the notion that the human bone marrow may be the equivalent of the avian Bursa of Fabricius (37). McMillan and Longmire (personal communication) have in addition recently demonstrated that human bone marrow cells in short-term culture are able to secrete immunoglobulin. The data presented here and other work in progress (Bell, D. A., McMillan, R., and Longmire, R. L., manuscript in preparation) further indicate that human bone marrow is enriched in specific antibody-producing cells. Mellbye has shown similar enrichment of PFC in the bone marrow of immunized mice (38). It would be premature however, to generalize that the bone marrow is the mammalian equivalent of the bursa. The techniques described here offer a new approach to studying antibody production in SLE in humans and NZB/W mice. It is hoped that future studies will allow quantitative comparisons to be made between the magnitude of the
302 antibody response this disease.
BELL
measured
ET
AL.
in this way,
and immunopathologic
events
in
ACKNOWLEDGMENTS We are indebted to Miss Nancy Erhardt and Mrs. Karen Hopper for skillful technical assistance, and to Mrs. Pat Curl for preparing the manuscript. We also thank Dr. John Johnson for making a number of helpful suggestions and reviewing the manuscript. REFERENCES 1. AL~rson-, A. C., DENMAN, A. M., AND BERNES, R. D., Z,~wr,l 2, 135, 1971. 2. FUDENBERG, H. H. Amer. J. Med. 51,3,295, 1971. 3. DACIE, J. V., “The Hemolytic Anaemias, Congenital and Acquired,” Grune and Stratton, New York, 1960. 4. PIROFSKY, B., “Auto Immunity and the Auto Immune Hemolytic Anaemias,” Baltimore, 1969. 5. DE VRIES, M. J., AND HIJMANS, W., Immunology 12, 179, 1967. 6. SEEGAL, B. C., ACCINNI, L., ANDRE& G. A., BEISER, S. M.. CIIRISTIAN, C. L., ERLANGEI(, B. F. AND Hsu, K. C., 1. Erp. Med. 130, 203, 1969. 7. TONIETTI, G., OLDSTONE, M. B. A., AND DIXON, F. J.. J. Exp. Med. 132, 89, 1970. 8. ANDRES, G. A., ACCINNI, L. BEISER, S. M.. CHRISTL~P;, C. L. CANOTTI, G. A., ERLAN(;~:H, B. F., Hsu, K. C., AND SEEGAL, B. C., J, C/in. Zwest. 49, 2106, 1970. 9. BARBU, E., SELIG~MAN, M., AND JOLY, M. Ann. Inst. Pasteur (Paris) 99, 695, 1960. 10. STOLLAR, B. D., LEVINE, L., LEHRER, H.,I., AND VAN VANUKIS, H., Biochem. B~o)J&s. Actu 61, 7, 1962. 11. ARANA, R., AND SELIGMAN, M. J. C/in. Incest. 46, 1867, 1967. 12. KOFFLER, D., CARR, R., AGNELLO, V., THOBURN, R.. AND KUNKEL, H. C:., J. Exp. Med. 134. 294, 1971. 13. SELIGMAK, M., AND ARANA, R., In “Nucleic Acids in Immunology” (0. J. Plescia and W. Braun, Eds.), Springer-Verlag, New York, 1968. 14. LAMBERT, P. H., AND DIXON, F. J., J. Ex~J. Med. 127, 507, 1968. 15. KOFFLER, D., SCHUR, P. H., AND KUNI
ANTI
31. 32. 33. 34. 35.
36. 37. 38.
DNA
ANTIBODY-PRODUCING
CELLS
303
SCHUR, P. H., MOROZ, L. A., AND KUNKEL, H. G., Immunochemistry 4,447, 1967. SHARP, G. C., IRVIN, W. S., LAROQUE, L. R., VELEZ, C., DALY, V., KAISER, A. D., AND HOLLMAN, H. R.,J. Clin. Inoest. 50,350, 1971. KOFFLER, D., AGNELLO, V., THOBURN, R. AND KUNKEL, H. G., Fed. Proc. 31, 661, 1972. HUGHES, G. R. V., COHEN, S. E., LIGHTFOOT, R. W., MELTZER, J. I., AND CHRISTIAN, C. L., Arthr. Rheum. 14, 259, 1971. DIXON, F. J. TONIETTI, G., OLDSTONE, M. B. A., AOKI, T., AND MCCONAHEY, P. J., In 11th Int. Immunopathology Symposium (P. A. Miescher, Ed.), Grune and Stratton, New York, 1971. STOLLER, D., AND SANDBERG, A. L., J. Immunol. 96,755, 1966. ABDOU, N. I., AND ABDOU, N. L., Science 175,446, 1972. MELLBYE, 0. J., Intern. Arch. Allergy Appl. lmmunol. 40, 249, 1971.
IhNUNOLOGY
AND
I~~~~L’SOPATHOLO(;~
1. 293-303
(1973)
Anti DNA Antibody Production by Lymphoid Cells of NZB/w Mice and Human Systemic lupus Erythematosus (SLE)’
Single-stranded DNA (S-DNA) coupled to sheep erythrocytes by the chromic chloride method was used to detect antibody-producing cells by a standard hemolytic plaque assay (PFC), and antigen sensitive rosette-forming cells (RFC) by the technique of immunocytoadherence. The spleens and other lymphoid tissue oft?l9-month-old NZB/W F, female mice were studied. The spleens of these mice showed anti-S-DNA antibody-producing cells, which reached a peak in the 7-9month-old mice, and correlated directly with the magnitude of the serum antis-DNA antibody detected by the Farr assay. Shrdies of human lymphoid cells from bone marrow and peripheral blood of patients with systemic lupus crythematosus (SI,E) also revealed significant numbers of anti-S-DNA PFC’s.
There is growing interest in the possibility that some autoimmune diseases may develop among those individuals who have an underlying genetic or acquired abnormality in lymphoid cell function (2-4). The nature of this underlying disorder(s) has not been established, although a number of different observations have suggested that an abnormality of thymus or thymus-derived cells may be involved (l-5). Systemic lupus erythematosus (SLE) represents a prototype autoimmune disorder in which a suitable animal model, the NZB mouse, is available for study. The development of techniques useful for study of the spontaneous immune responses to self antigens in these mice should also be useful in the study of such immune responses in humans with SLE. We have developed a reproducible system for studying those aspects of lymphoid cell function represented by plaque-forming cells and rosetteforming cells to single-stranded deoxyribonucleic acid (S-DNA). These techniques offer the opportunity of quantitating numbers of both antibodyproducing cells (PFC), and antigen sensitive cells (RFC) reactive to a welldefined antigen. The response to S-DNA was chosen since (1) this antigen is chemically well defined and readily available in highly purified form, (2) antibodies to S-DNA are present in a large proportion of both NZB/W mice (6,7) ’ This study was supported by NIH Grant AM 1511802. * Medical Research Council of Canada, Fellow, Division Research Foundation. Address after July 1, 1972: Department Ontario, London, Ontario, Canada. n Present address: V. A. Hospital, Minneapolis, Minnrsota. 293 Copyright All rights
@ 1973 hy Academic Press, Inc. of reproduction in any form reserved.
of Rheumatology, of Medicine,
Scripps University
Clinic and of Western
294
HELL
ET
.4L.
and patients with SLE (8-11,22), (3) a number of studies indicated that this is an important potential immunogen in man (12,13), and (4) antibody-antigen complexes involving DNA have been incriminated in the pathogenesis of both NZB/W mice (6,14) and humans with SLE (8,15). Although a number of studies in both humans and mice with SLE have examined serum or tissue fixed anti DNA antibody, no direct studies of the cellular mechanisms supporting the antibody response to this antigen have been reported. Such an approach should allow a direct study of factors effecting production of antibody to DNA both in z:iuo and in Gitro in these autoimmune disorders. In this report we show that S-DNA coupled to sheep erythrocytes (SRBC) may be used in a standard PFC assay to quantitate anti S-DNA antibodysecreting cells in the spleens of NZB/W mice. The same indicator SRBC’s can be used to detect a larger population of spleen cells in these mice which specifically bind to S-DNA, but do not secrete detectable quantities of complement-fixing antibody. Preliminary studies of these data have been presented previously (16). These techniques have been applied to detect antibody-secreting cells in humans with SLE, and a preliminary report of these data are presented here. MATERIALS
AND
METHODS
Animals. NZB/W F, female mice were provided by Dr. Frank Dixon, Scripps Clinic and Research Foundation. These mice were obtained from mating NZB/Bl females with NZW/Lac males, and were obtained from the colony of mice that have been used in previous studies from this institution (7, 14,17). Normal BDF, male mice (C57 B1/6 female X DBA/2 male) were obtained commercially (Jackson Laboratories, Bar Harbor, ME). Plaque-forming cell JPFC) cissay. Plaque-forming cells from spleens or other lymphoid cells of mice or humans were developed by a modification of the standard Jeme plaque assay in gel (18). Normal or DNA coupled SRBC (Colorado Serum Company) were mixed with single cell suspensions of leukocytes in melted 0.4% agarose, then poured onto microscope slides. The slides were incubated in a moist chamber for 60 min at 37“C, then exposed to 10% guinea pig complement (absorbed with SRBC) with or without facilitating serum (see later). The slides were then incubated again for 60 mm at 37°C then at 4°C overnight and read with the aid of a hand lens. In experiments designed to detect 7S PFC, goat anti-mouse 7SyG serum absorbed with SRBC was used. Two different sera were used. One serum was obtained commerically, and a second was a gift from Dr. Bruce Clinton, Scripps Clinic and Research Foundation. These sera were equally capable of maximally facilitating the development of 7S anti SRBC PFC in immunized normal (BDF,) mice. In studies designed to detect 7S PFC of human lymphoid origin, rabbit anti human 7SrG serum absorbed with SRBC was used, This serum was obtained by hyperimmunizing rabbits with human Cohn Fraction II in cornplete Freund’s adjuvant. Rosette-forming ceE1(RX) c~ssay. Rosette-forming cells were developed by
ANTI
DNA
ANTIBODY-PRODUCING
CELLS
295
a modification of the centrifugation suspension technique of McConnel et ul. (19). For these studies the normal or DNA coupled SRBC used for plaquing were mixed in a 20: 1 ratio with washed mouse leukocytes (5-10 x lo6 nucleated cells/ml). This mixture was centrifuged at 1OOg for 15 min at 4°C incubated a further 30 min at 4”C, then gently resuspended in Balanced salt solution (BSS) (20) to a convenient concentration for counting; 3000-5000 nucleated cells were scanned for leukocytes with at least four adherent erythrocytes. Duplicate counts were made in each instance and the average number of RFC/106 nucleated cells calculated. In some instances, the rosettes were stained with Giemsa on microscope slides in order to study the morphologic appearance of rosetting cells. Coupling of DNA to sheep erythrocytes. Calf thymus DNA (Worthington Biochemical Corp., Freehold, N. J.) was rendered single stranded by heating to 100°C for 12 min, followed by rapid cooling in an ice bath. One milliliter of this single-stranded DNA at a concentration of 500 pglml was then mixed with 0.1 ml of 2% chromic chloride (J. T. Baker Chem. Co., Phillipsburg, N. J.), and incubated at ambient temperature with 0.2 ml of 50% washed sheep erythrocytes. The coupled sheep erythrocytes were then thoroughly washed in phosphate buffered saline (PBS) pH 7.3 and prepared as a 7% suspension in BSS for plaquing or stored at 4°C until required. S-DNA was detected on these cells by passive hemagglutination or passive immune hemolysis with rabbit anti S-DNA serum absorbed with SRBC. Derivatization was found to be constant by these means among different cell preparations, and such cells remained stable at 4°C for at least 6 days. Serum anti S-DNA antibody. The serum of both NZB/W and BDF, mice were examined for the presence of anti S-DNA antibody by the sensitive Farr assay. S-DNA used in these studies was sonicated prior to labeling. Sonication was performed with a Branson Sonifier (model S-125 Branson Instruments Inc., Danbury, Conn.) 20,000 kHz at 2.5 A for 2 min. Two micrograms of sonicated single-stranded calf thymus DNA labeled passively with tritiated actinomycin D (4 &i of tritiated actinomycin D per mg of DNA) as previously described, (21,22), was mixed with a 1: 10 dilution of heat inactived test serum for 60 min at 37°C then at 4°C for 18 hr. The mixture was then precipitated by the addition of saturated (NH,),SO, to a final concentration (vol : vol) of 50%, and the precipitate washed with 50% saturated (NH&SO,. The average number of counts per minute of the labeled S-DNA precipitated by each serum was calculated and expressed as the percentage of antigen binding capacity of the serum (ABC). RESULTS The numbers of PFC and RFC in normal BDF, mice are summarized in Table 1. Few anti S-DNA PFC or RFC were detected in these normal mice. Similar studies of PFC and RFC in NZB/W mice demonstrate that a significantly higher number of anti S-DYA reactive cells exist in the spleens of these mice (Table 2). The NZB/W rn; :e showed a range of from 16 to 452 direct (19s) PFC and from 13,000 to 85,000 RFC/106 nucleated spleen cells, the
296
BELL
ET
AL.
TABLE
1
ANTI S-DNA PFC AND RFC IN SPLEENS OF NORMAL (BDF,) MICE” Direct
RFC/lO”
PFC/lO’
“IO(l)” 2 (1)
Av
“2 7.7 7 4.4 7.9 7
4 (1) ‘6.2 (1.5) ‘1 (1) “1 (1) l(l) 1 (1) d3.2 (1.0)
(1.5) (7.7) (7) (9.6) (7.9) (7)
lF(‘5.6, “6.9 (6)
a Data represent PFC or RFC values in pools of two mice per pool Ir Numbers in parentheses indicate background PFC or RFC values values. c Data represent PFC or RFC from single spleens. d Arithmetic mean. e Not tested.
TABLE
except where indicated vs normal sheep RBC
2
ANTI S-DNA PFC AND RFC IN SPLEENS OF UNIMMUNIZED B/W MICE
Age ho) 6 6 6 6 7 7 8 9 9 9 9 9 9 10 10 10 10
PFC/lO” Direct
Anti Indirect
69 (l)*
20 (1) 21 (0) 22 (0) 259 452 253 109 132 214
(1) (4) (2) (1) (4) (2) 16 (1) 20 (1) 168 (2)
83 (59 74 0) 34 (10) 94 (10)
q % ABC is % of labeled the presence of 50% (NH,), b Numbers in parentheses e Not tested.
RFC/
Spleen
% ,4BC’
lo3
wt
(mgl -’
0 0 0 0
-
-( -
24 85 53 28 25 33 36
0 11 18
-
S-DNA SO,. denote
Serum S-DNA
-
(11) (10)
-.
(7) (7) (5) (14)
553
(8) -
-_ 239 114 301 963
82 (88) -
13 (6.3) 55 (3.8) precipitated
by a 1:lO
background
PFC
dilution
or RFC
of heat
vs normal
inactivated
SRBC.
serum
in
ANTI
DNA
ANTIBODY-PRODUCING
297
CELLS
latter representing 6,700 to 75,000 anti S-DNA RFC. The number of anti S-DNA PFC correlated directly with the amount of serum anti S-DNA detected in the Farr assay. The quantity of antibody to S-DNA detected by both of these procedures appeared to be higher among animals 7-9 months of age, although considerable variability existed even among animals of the same age group. None of the animals studied were moribund at the time they were sacrificed. These mice also showed a wide variability in the number of anti S-DNA RFC and spleen weights, neither of which correlated with age or S-DNA antibody production. Attempts to demonstrate 7s anti S-DNA PFC by the addition of varying dilutions of facilitating serum usually resulted in either no facilitation or actual suppression of tota PFC. Only two of the mice studied had detectable 7s PFC demonstrated by this technique. The sera used for these studies were readily capable of facilitating anti SRBC PFC in BDF, mice primed with SRBC. Table 4 summarizes the result of a number of experiments designed to show specificity for S-DNA in the rosette-forming cell assay system. In these experiments spleen cells of individual NZB/W mice were preincubated for 60 min at 4°C with varying concentrations of S-DNA, native DNA, or human serum albumin. These spleen cells were then mixed with normal or S-DNA coupled SRBC in the usual way. Inhibition of anti S-DNA RFC with S-DNA proved more difficult than inhbition of anti S-DNA PFC (Table 3). At the concen-
INHIBITION PFC S-DNA Expt’ 1
2
Av inhib (%) S-DNA PFC Inhib (%) SRBC PFC*
TABLE 3 OF ANTI S-DNA WITH S-DNA Inhibitor
(pg)
Nil
.OOl
.Ol
1
245 119 136 112 38 121 140 141 82
112 71 89 99 -
40 31 8 15 -
-c
-
-
-
4 23 16 12 37
0 7 8 7 27
-
35
82
82
94
-
-
0
6%
-e
n Data represent PFC/lOG nucleated cells in spleens of eight individual separate experiments, and are the average of replicate determinations. * Data represent av % inhibition of direct anti SRBC PFC in three BDF, previously with 0.2 ml 10% SRBC iv. c Not tested.
5
B/W mice
mice
in two
primed
4 days
BELL
298
SPLEEN
ET
AL.
TABLE 4 INHIBITION OF ANTI S-DNA RFC IN NZB/W CELLS WITH S-DNA, N-DNA, AND HUMAN SERUM
ALLWMIX~
Inhibitor
S-DNA (/.d Nil
.Ol
6 8.3 6.5 8.3 27 36 28 30
28 -
34 55
34 -
82
75
Av. 9% Inhib
.05
1
1.3 2.3 -
3
3.3 3.1 2 6 12 10 10 20 24
80
69
N DNA 5
10
2.1 1.3 -
-
-
75
(wg)
.I
HSA 1
5 7
100
5 6 6.8 6.2
1 6 12 3 4 2 9
-. -
-
85
17
17
!cLg)
16 8 -. .-
2
u Data represent net number of RFC/10” vs S-DNA after preincubation of spleen cells from individual mice with denatured DNA (S-DNA), native DNA (NDNA) or human serum albumin (HSA). Numbers represent the average of duplicate determinations.
trations tested, S-DNA showed significantly more inhibition (69-85s) of anti S-DNA RFC than either “native” DNA (17%) or human serum albumin. Distribution of PFC and RFC in lymphoid cells of NZBIW mice. Table 5 shows preliminary studies comparing the distribution of PFC and RFC values in pooled peripheral lymph nodes, spleen, and thymus of two unimmunized NZB/W mice (age 6 mo) compared with three BDF, mice primed 5 days previously with 2 x lo* SRBC intravenously. Although the anti S-DNA response of these B/W mice was relativeIy low, it is clear that spleen cells
DISTRIBUTION NZB/W
NZB/W NZB/W BDF,
TABLE 5 OF PFC AND RFC MICE AND IMMUNIZED
Spleen
PFC/lOG Nodes
21” 7 1610*
1.5 1 2.5
IX UNIMMUNIZED BDF, Mice
Thymus
Spleen
1.5 0 4.5
6.4 6.4 39.
RFC/l@’ Nodes
Thi
0 .2 1.6
.6 .5 1.2 _-
u Net number of anti S-DNA PFC/lOe or RFC/103 nucleated cells. b Direct anti-SRBC PFC/lOB or RFC/lO” nucleated cells in BDF, with SRBC (pooled tissues of three mice).
mice
5 days
after
priming
ANTI
DNA
ANTIBODY-PRODUCING
TABLE
299
CELLS
6
ANTI S-DNA PFC IN HUMAN BONE MARROW AND PERIPHERAL BLOOD LEUKOCYTES PFC/lOs Bone Pt.
Diagnosis
T.R.
SLE
L.C.
SLE
L.F. H.R. D.S. E.B.
SLE SLE SLE SLE Normald
Prednisone Nil 60mg
marrowa~*
Direct
Indirect
4 (0) 251( 105) 2 (0) 8 (3) 0 -
Peripheral Direct
> 1000
(0)
Indirect
0 0 -
5 (0) 0 108 (0) 0 0 0 0
200-400 0 0 0 10 (0) 0 0
-
0
0
0 200-400
(0)
blood* Disease (0)
activity -
++ i +++ 2 ++ * -
a From iliac crest. * Erythrocytes removed by NH&l lysis and cells washed three times in balanced salt solution. c Background PFC vs normal SRBC values are in brackets. d Peripheral blood leukocytes from three normal donors studied as controls in three different experiments with SLE leukocytes.
make the largest contribution to the total number of direct PFC and RFC in both the NZB/W mice and the SRBC primed BDFl mice. Anti S-DNA PFC in human lymphoid cells. Table 6 outlines our experience in employing the foregoing techniques to study antibody-secreting cells in human SLE. In these studies leukocytes from bone marrow and/or peripheral blood of patients with SLE were plated in the usual way with normal or S-DNA coupled sheep erythrocytes and the number of PFC per lo6 nucleated cells calculated. As shown in this table, the bone marrow cells of four of five patients had detectable numbers of PFC above background and in three of these patients large numbers of anti S-DNA PFC were seen. Two of these patients had, in addition, significant numbers of S-DNA PFC in their peripheral blood, These peripheral blood PFC were not detectable in the three normal donors or in the SLE patients after initiation of high dose corticosteroid therapy. The table indicates that both 19s and 7s PFC were identified in these patients, which contrasts with our observations showing a paucity of 7s anti S-DNA PFC in the NZB/W mice. The finding of significant numbers of 7s PFC among some of these human lymphoid cells, indicates that our inability to detect such 7s PFC in the NZB/W mice is probably not related to inadequate derivatization of SRBC with S-DNA. DISCUSSION The data reported here show that sheep erythrocytes derivatized with S-DNA by the chromic chloride method provide convenient target cells for
300
BELL
ET
AL.
studying specific antibody-producing cells and antigen sensitive cells in both humans and NZB mice with systemic lupus erythematosus. No similar studies of S-DNA antibody-producing cells have previously been documented in either mice or humans with SLE. In addition to the obvious interest in the distribution of antibody-forming cells among various compartments of the lymphoid system, there are a number of advantages to studying antibody production at the cellular level. The PFC technique avoids the difficulty presented by antibody being masked by complexing in the circulation with antigen, a phenomenon presumed by others (26,33) to have made it impossible for them to demonstrate antibody and antigen simultaneously in the circulation of some SLE patients. The release of DNA into the circulation following a variety of stresses and corticosteroid therapy and consequent sudden fall in detectable serum anti DNA antibody in patients with SLE (34), may be a further example of this phenomenon. These factors place a limitation on the interpretation of changes in serum anti DNA antibody levels during the course of this disease. The PFC technique on the other hand, allows day by day evaluation of antibody production, which may vary with disease or as a part of the therapeutic regimen. The technique may also theoretically allow one to obtain information on the affinity of antibody molecules being secreted at any given time. Although affinity measurements may be obtained through an examination of serum antibody itself, the masking factors already mentioned may not allow study of those antibody molecules of highest affinity. Such information may be of value in attempting to study the effects of agents designed to manipulate the immune response. The use of PFC and RFC detection should provide a means for assessing tolerance to nucleic acid antigens in experimental animals, and can be expected to provide interesting data on the effects of virus infection on antibody responses. A number of recent studies by Oldstone and collaborators (7,17) have shown that some viruses may accelerate and others depress certain features of the disease in NZB/W mice, including the level of serum anti S-DNA antibody. The mechanism leading to these effects could be profitably studied at the cellular level, in ciao or in citro, since it is presently not known whether viruses alter the form and/or immunologically effective concentration of host nucleic acid or other antigens, or whether they exert their effects by modifying the activity of cells destined to, or already in the process of secreting antibody. These studies reported here, show that anti-SDNA PFC in the spleens of these NZB/W mice peak at 7-9 months of age, a time which corresponds to the period of greatest mortality from glomerulonephritis, reported in these mice (35). Those mice which had survived beyond this period had relatively fewer anti-SDNA PFC. A surprising aspect of the immune response observed in these chronically ill mice is the relative paucity of 7S anti-SDNA PFC. Since others have reported that the predominant class of antibodies detectable in the renal glomerulus of NZB/W mice are in the IgG class, this observation of a predominance of 19s antibody producing spleen cells will require further
ANTI
DNA
ANTIBODY-PRODUCING
500400300cp 0 r. z z .-P i
. .
.
200-
301
CELLS
.
0.
.
.
loo-
. .
40-
.
% Normal
r;,
[BDFl)
NZB/WFl 20-
?*
Miceo Mice*
l
B I 10 20 30 Serum % S.DNA Binding FK.
40 Capacity
50 [ABC]
I 60
1.
study. Our ability to detect 7s anti-SDNA PFC in human lymphoid cells using the same derivatized SRBC, however, make it seem unlikely that this observation is merely related to technical factors in our assay system. While the studies reported here demonstrate a general direct correlation between serum antibody levels, and splenic PFC, no such correlation was seen with the larger population of splenic RFC and either serum antibody or PFC. This observation is consistent with the notion that the anti-SDNA rosetting system used here may be measuring a heterogeneous population of antigen reactive cells. Studies of the exact nature of this population of SNDA reactive cells are needed, with particular regard to their possible origin from bone marrow and/or thymus. The data on antibody-secreting cells in the peripheral blood and bone marrow of our SLE patients are somewhat preliminary, since only a few patients have been studied. Surprisingly no published studies have been made of the antibody producing potential of human bone marrow or human peripheral blood leukocytes using the techniques reported here. Abdou and Abdou have recently presented evidence supporting the notion that the human bone marrow may be the equivalent of the avian Bursa of Fabricius (37). McMillan and Longmire (personal communication) have in addition recently demonstrated that human bone marrow cells in short-term culture are able to secrete immunoglobulin. The data presented here and other work in progress (Bell, D. A., McMillan, R., and Longmire, R. L., manuscript in preparation) further indicate that human bone marrow is enriched in specific antibody-producing cells. Mellbye has shown similar enrichment of PFC in the bone marrow of immunized mice (38). It would be premature however, to generalize that the bone marrow is the mammalian equivalent of the bursa. The techniques described here offer a new approach to studying antibody production in SLE in humans and NZB/W mice. It is hoped that future studies will allow quantitative comparisons to be made between the magnitude of the
302 antibody response this disease.
BELL
measured
ET
AL.
in this way,
and immunopathologic
events
in
ACKNOWLEDGMENTS We are indebted to Miss Nancy Erhardt and Mrs. Karen Hopper for skillful technical assistance, and to Mrs. Pat Curl for preparing the manuscript. We also thank Dr. John Johnson for making a number of helpful suggestions and reviewing the manuscript. REFERENCES 1. AL~rson-, A. C., DENMAN, A. M., AND BERNES, R. D., Z,~wr,l 2, 135, 1971. 2. FUDENBERG, H. H. Amer. J. Med. 51,3,295, 1971. 3. DACIE, J. V., “The Hemolytic Anaemias, Congenital and Acquired,” Grune and Stratton, New York, 1960. 4. PIROFSKY, B., “Auto Immunity and the Auto Immune Hemolytic Anaemias,” Baltimore, 1969. 5. DE VRIES, M. J., AND HIJMANS, W., Immunology 12, 179, 1967. 6. SEEGAL, B. C., ACCINNI, L., ANDRE& G. A., BEISER, S. M.. CIIRISTIAN, C. L., ERLANGEI(, B. F. AND Hsu, K. C., 1. Erp. Med. 130, 203, 1969. 7. TONIETTI, G., OLDSTONE, M. B. A., AND DIXON, F. J.. J. Exp. Med. 132, 89, 1970. 8. ANDRES, G. A., ACCINNI, L. BEISER, S. M.. CHRISTL~P;, C. L. CANOTTI, G. A., ERLAN(;~:H, B. F., Hsu, K. C., AND SEEGAL, B. C., J, C/in. Zwest. 49, 2106, 1970. 9. BARBU, E., SELIG~MAN, M., AND JOLY, M. Ann. Inst. Pasteur (Paris) 99, 695, 1960. 10. STOLLAR, B. D., LEVINE, L., LEHRER, H.,I., AND VAN VANUKIS, H., Biochem. B~o)J&s. Actu 61, 7, 1962. 11. ARANA, R., AND SELIGMAN, M. J. C/in. Incest. 46, 1867, 1967. 12. KOFFLER, D., CARR, R., AGNELLO, V., THOBURN, R.. AND KUNKEL, H. C:., J. Exp. Med. 134. 294, 1971. 13. SELIGMAK, M., AND ARANA, R., In “Nucleic Acids in Immunology” (0. J. Plescia and W. Braun, Eds.), Springer-Verlag, New York, 1968. 14. LAMBERT, P. H., AND DIXON, F. J., J. Ex~J. Med. 127, 507, 1968. 15. KOFFLER, D., SCHUR, P. H., AND KUNI
ANTI
31. 32. 33. 34. 35.
36. 37. 38.
DNA
ANTIBODY-PRODUCING
CELLS
303
SCHUR, P. H., MOROZ, L. A., AND KUNKEL, H. G., Immunochemistry 4,447, 1967. SHARP, G. C., IRVIN, W. S., LAROQUE, L. R., VELEZ, C., DALY, V., KAISER, A. D., AND HOLLMAN, H. R.,J. Clin. Inoest. 50,350, 1971. KOFFLER, D., AGNELLO, V., THOBURN, R. AND KUNKEL, H. G., Fed. Proc. 31, 661, 1972. HUGHES, G. R. V., COHEN, S. E., LIGHTFOOT, R. W., MELTZER, J. I., AND CHRISTIAN, C. L., Arthr. Rheum. 14, 259, 1971. DIXON, F. J. TONIETTI, G., OLDSTONE, M. B. A., AOKI, T., AND MCCONAHEY, P. J., In 11th Int. Immunopathology Symposium (P. A. Miescher, Ed.), Grune and Stratton, New York, 1971. STOLLER, D., AND SANDBERG, A. L., J. Immunol. 96,755, 1966. ABDOU, N. I., AND ABDOU, N. L., Science 175,446, 1972. MELLBYE, 0. J., Intern. Arch. Allergy Appl. lmmunol. 40, 249, 1971.