Autoimmune NZB Mice (Immunoglobulin Genetics/Igd/Hematopoiesis/Colony-Stimulating Factors/Prostaglandins) PAUL W

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Proc. Nati. Acad. Sci. USA Vol. 76, No. 7, pp. 3464-3468, July 1979 Immunology Abnormalities in clonable B lymphocytes and myeloid progenitors in autoimmune NZB mice (immunoglobulin genetics/IgD/hematopoiesis/colony-stimulating factors/prostaglandins) PAUL W. KINCADE, GRACE LEE, GABRIEL FERNANDES, MALCOLM A. S. MOORE, NEIL WILLIAMS, AND ROBERT A. GOOD Sloan-Kettering Institute for Cancer Research, 145 Boston Post Road, Rye, New York 10580 Contributed by Robert A. Good, April 6, 1979

ABSTRACT Cloning procedures were used to study B ability of precursor cells of NZB mice to form myeloid colonies lymphocytes and progenitors of granulocytes and macrophages in response to a particular stimulus in vitro. Both of these were in NZB mice. Numbers of B cells that were detected in sheep characteristic of young NZB mice of both sexes and were also erythrocyte-containing semisolid cultures were only slightly in F1 of NZB mice mated with elevated in NZB tissues, and these were normally sensitive to partially expressed progeny inhibition by anti-,u or anti-b antibodies or prostaglandin E. NZW or CBA strain mice. However, NZB mice rapidly developed large numbers of B cells that could be cloned in the presence of lipopolysaccharide, and MATERIALS AND METHODS these included unusual anti-,u resistant cells. Numbers of myeloid precursors in NZB bone marrow that were responsive to Animals. Allotype congenic C57BL/6.Ige mice were donated colony-stimulating activity in L-cell conditioned medium or by Noel Warner. NZB/Umc, CBA/H, (NZB X NZW)FI, (NZB endotoxin serum were at least normal, but at all ages granulo- X CBA)F1, and NZW/Umc were all produced at Sloan-Ket- cyte-macrophage precursors were poor responders in cultures tering from breeding stock obtained from the University of stimulated by WEHI-3 cell conditioned medium. Almost no Minnesota. colonies were elicited in NZB cultures with a colony-stimulating Cell Cultures. McCoy's modified 5a medium containing 15% activity moiety from WEHI-3 cells. Prostaglandin sensitivity respect to as- of myeloid precursors from NZB and CBA mice was also dif- fetal calf serum and enriched with glutamine, ferent. Codominant genetic control of these abnormalities was paragine, serine, pyruvate, and amino acids was used for all suggested by their partial expression in F1 hybrid NZB X CBA semisolid agar cultures as described (12-15). B lymphocytes and NZB X NZW mice. NZB mice expressed an unexpected IgD were cloned in the presence of 50 ,gM 2-mercaptoethanol and allotype allele. either 25 jig of Salmonella typhosa endotoxin (lipopolysaccharide; LPS) per ml or 0.1% washed sheep erythrocytes Abnormalities have been found in or attributed to T cells, (SRBC). The importance of using LPS or SRBC for achieving thymic epithelium, B cells, macrophages, and stem cells in reproducible colony numbers and a linear dose-response curve autoimmune NZB mice, and they produce high levels of is discussed in previous reports (12, 14). Cultures were examined xenotropic virus (1-6). The relevance of each of these abnor- with a dissecting microscope for the presence of colonies (ag- malities to the development of the hemolytic anemia in these gregates of >20 cells) after 6 days of incubation at 370C in a mice is not clear, but genetic studies tend to minimize the role fully humidified atmosphere of 7% CO2 in air. Granulocytic of viruses and certain hemopoietic abnormalities in the etiology macrophage progenitors were similarly cloned in the absence of disease (5, 6). A possible temporal sequence is suggested by of 2-mercaptoethanol in cultures containing an appropriate findings that B cells of NZB mice are polyclonally activated by source of colony-stimulating activity (CSA). 1 week of age, and autoantibodies with preference for sup- Granulocyte-Macrophage CSA. Stimuli used for cloning pressor T cells are detectable early in adult life (7-10). A pri- neutrophil and macrophage colonies were concentrates of mary B-cell abnormality or defects that are expressed at the conditioned medium from WEHI-3 myelomonocytic leukemia B-cell level could thus predispose to subsequent aberrant T-cell cells (WEHI-3-M) and L cells (LCCM). Both cell lines were function. On the other hand, production of antierythrocyte propagated in 75-cm2 plastic flasks in McCoy's 5a medium autoantibody may occur in the absence of significant numbers containing 2.0% fetal calf serum and 50,uM 2-mercaptoethanol. of T cells, and potential for autoimmunity is transferrable with Medium was harvested at 3- to 4-day intervals and concentrated fetal liver or bone marrow cells from NZB mice (3, 11). Some 5-fold by Amicon (Lexington, MA) UM 10 ultrafiltration. gene products influencing manifestation of autoimmunity Serum-free medium conditioned by WEHI-3 cells for 3-4 days might therefore be expressed in pre-B cells, B cells, or non- was concentrated and equilibrated with 0.02 M Na phosphate lymphoid cells capable of activating B cells. Limiting dilution buffer, pH 8.0, and then passed through DEAE-Sephadex as cloning techniques are available for enumerating and charac- described (16). This material will be subsequently referred to terizing several hemopoietic precursors as well as lymphocytes, as semipurified WEHI-3 CSA. A fourth source of CSA was and we propose that these can be of value for identifying in- serum from CBA or NZB mice taken 3 hr after an intravenous trinsic defects in various cell types and for following changes injection of 5 ,ig of LPS endotoxin. Prostaglandin E (PGE) was in these with progression of disease. Thus far, we have found donated by John Pike (Upjohn), and this was stored at 1 mM in that particular culture conditions reveal an exceptional category ethanol at -70°C. of B lymphocytes in NZB lymphoid tissues, which, unlike B cells Anti-Immunoglobulin Antibodies. Anti-IgM antibodies from many other strains, proliferate in the presence of anti-ji were collected from goat anti-mouse myeloma M 104E (ji, X) antibody. In addition, a severe abnormality was detected in the serum on an absorbent of HPC-76 (ji, K) and rendered specific The publication costs of this article were defrayed in part by page Abbreviations: CSA, colony-stimulating activities; ES, serum from mice charge payment. This article must therefore be hereby marked "ad- injected with endotoxin; LCCM, medium conditioned by L cells; LPS, vertisement" in accordance with 18 U. S. C. §1734 solely to indicate lipopolysaccharide; PGE, prostaglandin E; SRBC, sheep erythrocytes; this fact. WEHI-3-M, medium conditioned by WEHI-3 cells. 3464 Downloaded by guest on September 28, 2021 Immunology: Kincade et al. Proc. Natl. Acad. Sci. USA 76 (1979) 3465 for it-chains by repeated passage over HOP-1 (y2a, A), UPC 10 lo" (QY2a, K), and MPC 11 ('Y2b, K) immunoabsorbents. Goat anti-K I antibodies were prepared by immunizing with F(ab')2 frag- c 6ea a, ments of MOPC-460 (a, K), eluting the resulting antibodies a, * a from the HPC 76 column, and then crossabsorbing on the HOP 'a 0 0o

1 column. Polyvalent anti-Ig antibodies from rabbit anti-TEPC 0 0 ~~8 183 (w, K) serum were eluted from a protein A-Sepharose CD e, (Pharmacia) absorbent. Ascites or serum from mice bearing the H6/31 anti-IgD allotype hybridoma (17) was purchased from Sera Labs (Crawley Down, Sussex, England). These reagents specifically lysed clonable B cells from C57BL/6J tissues at a 1:50 dilution in the presence of rabbit complement (1:12) and inhibited B-cell cloning when added directly to the cultures at

a 1:200 final concentration. Under the same conditions, cloning Ca of NZB or C57BL/6.Ige B cells was not significantly inhibited. 0) 0o Two other anti-IgD hybridomas produced at Stanford (18) were obtained from the Salk Institute Cell Distribution Center and maintained in RPMI 1640 medium. Culture supernates from 5 10 15 20'25 30 354045 50 the 11-6.3 hybrid cell line inhibited B-cell colony formation Age, weeks from C57BL/6 but not A/J, CBA/H, NZB, or C57BL/6.Ige FIG. 1. Clonable B lymphocytes in NZB (e) and CBA/H (0) mice at a 1:100 final dilution in the cultures. The pH of the spleens in cultures potentiated with LPS. Colony-forming units per 10-4.2 culture supernates was increased to 8.6 with concentrated spleen were calculated by multiplying numbers of total nucleated cells Tris, and the supernates were passed through the protein A- per spleen times the average incidence of colonies in four replicate Sepharose column. Approximately 3 ,tg of protein per ml of cultures of 2 X 104 cells. culture fluid was eluted from the column with glycine.HCl buffer, pH 2.8, and this was neutralized, concentrated, and that NZB B cells were completely resistant to H-6/31 hybri- dialyzed. This preparation contained only murine IgG2a by doma anti-6 antibody, whereas colony growth of A/J B cells was Ouchterlony analysis and was subsequently referred to as pure inhibited. These strains have been found to share alleles at all 10-4.2 anti-6. It was not cytotoxic for B cells with our rabbit other immunoglobulin allotype loci (21). Furthermore, A/J B complement but inhibited cloning of B cells from NZB, cells do not express the IgD specificity detected by the 10-4.2 C57BL.Ige, and CBA/H mice when added directly to the cul- hybridoma antibody, whereas both NZB and normal allotype tures. B cells of both A/J and C57BL/6 strains are negative for congenic C57BL/6.Ige strain mice do. C57BL/6.Ige mice were this specificity. A broader discussion of the expression of IgD derived from mating of NZB and C57BL/6 strains with sub- allotype alleles will be presented elsewhere (J. Goding, P. W. sequent backcrossing to C57BL, and BL/6.Ige mice share all Kincade, and L. A. Herzenberg, unpublished results). known Ig alleles with NZB mice (22, 23). These results suggest Depletion of B Lymphocytes. B cells from spleen, lymph that a recombination occurred such that alleles of the Ig-5 locus nodes, or bone marrow were depleted by adherence to anti-Ig differ in A/J and NZB mice. In several experiments with cul- coated petri dishes according to the method of Wysocki and tures of NZB cells, both the degree of colony inhibition and the Sato (19). Nonspecific cell loss was estimated by use of parallel amount of purified 10-4.2 hybridoma anti-6 antibody required uncoated dishes and by incubating allotype-negative cells in were similar to results obtained with other strains of mice (Fig. anti-6 coated dishes. Depletion of sIg+ cells always exceeded 2). The same result was obtained when heterologous rabbit 97% when anti-,t or anti-IgM coated dishes were used. anti-mouse IgD serum was used (data not shown). Sensitivity of Colony-Forming B Cells to Anti-,g Anti- RESULTS bodies. We reported that inclusion of divalent anti-Ai or anti-K Incidence of Clonable B Cells. When B cells are cultured antibodies in LPS- or SRBC-potentiated semisolid cultures al- in semisolid agar cultures in the presence of 2-mercaptoethanol most completely prevents colony formation (15). This has been and either LPS or SRBC, a linear relationship exists between true for cultures of B lymphocytes from CBA/H, CBA/J, the number of B cells present and the number of proliferating CBA/cum, C57BL/6, A/J, B6D2FI, BALB/c, C3H, and SJL clones (14). Numbers of clonable B cells in NZB tissues were mice. A significant proportion of the LPS-potentiated colony- markedly elevated relative to the numbers and proportions of forming cells in NZB tissues are unaffected by 25 ,tg of anti-,u these found in CBA mice. This was apparent in LPS-potentiated antibodies per ml. These unusual cells were detectable in spleen cultures of mice 1-50 weeks old (Fig. 1). The incidences of as early as 1 week after birth and reached maximal incidence colony-forming B cells in SRBC-potentiated cultures of CBA at around 17 weeks of age (Fig. 3). At this time they comprised and NZB tissues were usually comparable, and the total num- an average of 18%, 30%, and 41% of the LPS-potentiated bers of colony-forming cells per spleen were consistently high colonies in culture of NZB bone marrow, spleen, or lymph in NZB mice only after development of splenomegaly (not nodes, respectively (Table 1). Anti-,u antibodies inhibited colony shown). The numbers of colonies obtained with LPS alone or formation by greater than 99% in cultures of CBA tissues at all SRBC alone are not always equivalent with various normal ages tested. Inhibition of NZB colony formation was essentially B-cell populations, and approximately additive values are often complete when polyvalent anti-IgM (,, K) was added to the obtained when the two potentiators are used together (12, 14). cultures. Furthermore, an equivalent number of NZB and CBA This was also true for NZB cells (data not shown). spleen- or lymph node-clonable B cells adhered to petri dishes IgD Expression on NZB B Cells. A proportion of the col- coated with anti-,u or anti-IgM antibodies (data not shown). ony-forming B cells in tissues of normal adult mice are ex- Nonspecific cell loss with the dish depletion technique has al- tremely sensitive to anti-b antibodies (15). Others have found ways been less than 30% and is often less than 5%. This finding that the B cells of NZB mice have a low density of surface IgD suggests that the anti-,I resistant cells in NZB tissues are in fact relative to IgM (20), so we tested the effect of addition of anti-6 B cells and must express at least small amounts of surface IgM. antibodies to cultures of NZB cells. An unexpected finding was In contrast to the results with LPS-potentiated cultures, both Downloaded by guest on September 28, 2021 3466 Immunology: Kincade et al. Proc. Natl. Acad. Sci. USA 76 (1979) Table 1. Sensitivity of B lymphocyte colony formation to anti-p antibodies Mean no. of colonies ± SEM Medium alone - Anti-,p 0 NZB Bone marrow 801 i 147 145 + 51 Spleen 5417 + 601 1641 + 218 + 0 Lymph nodes 3103 + 192 1257 94 on CBA Bone marrow 655 + 56 1 ± 1 Spleen 1059 + 87 7 ± 3 0 + 0 Lymphnodes 475 + 172 1 1 Sensitivity was measured in LPS-potentiated cultures with or without 25,ug anti-p antibodies per ml. Five 17-week-old NZB mice ).0 are compared to the same number of 9-week-old CBA mice. Final concentration, gg/ml FIG. 2. Sensitivity of NZB (0) and normal allotype congenic studies from this laboratory have emphasized C57BL/6.Ige (0) B-lymphocyte colony formation to pure 10-4.2 hy- the regulatory bridoma anti-6 antibodies. Each point is the mean (±SEM) number potential of prostaglandins for lymphohemopoietic cells (24). of colonies in four replicate cultures. B-lymphocyte cultures of NZB and CBA cells prepared with LPS or SRBC were similarly sensitive to 0.01-50,tM synthetic prostaglandin E (PGE) (data not shown). However, NZB NZB and CBA B-lymphocyte colony formation in the presence colonies stimulated by WEHI-3 CSA were unusually sensitive of SRBC was completely inhibited by anti-A antibodies. to PGE, whereas their L-cell CSA-stimulated colonies were Granulocyte-Macrophage Progenitors. Nonlymphoid more resistant to PGE than were those of CBA controls (Fig. colony formation in semisolid cultures is dependent on CSA 5). obtainable from various sources. We employed WEHI-3-M for Heritability of NZB Abnormalities. In most of our experi- this purpose in the majority of our experiments. The incidence ments we have compared NZB to normal CBA/H mice. It is of WEHI-responsive progenitors was relatively constant at all important to establish whether the abnormal features of these in bone marrow and + autoimmune mice are common to the more closely related ages CBA averaged 162 8 colonies per normal NZW mice and whether they are expressed in F1 105 cultured cells in 23 determinations. NZB bone marrow was progeny of NZB parents. One such comparison is illustrated in markedly deficient in these cells at all stages and averaged only Fig. 6. NZW mice had more B cells that were anti-ti resistant 43 + 6 colonies per 105 cultured cells in 22 assays. The gross in LPS-potentiated cultures than CBA mice but far fewer than morphology of NZB colonies was abnormal in that the cells were NZB. Individual (NZB X NZW)F1 animals tended to have in- unusually dispersed. This defect in in vitro myelopoiesis was termediate numbers of abnormal B cells, and the same was even more apparent when a semipurified fraction of WEHI-3 found for hybrids of NZB and CBA. The incidence of WEHI-3 CSA was employed and almost no NZB colonies were stimu- CSA-responsive cells in NZW and CBA bone marrow were lated by it (Fig. 4). Cultures of equal part mixtures of NZB and similar as were their sensitivities to 0.1 MAM PGE (Fig. 7). Again, CBA bone marrow gave intermediate numbers of colonies with F1 offspring tended to have characteristics that reflected the this stimulator (data not shown). In striking contrast to these influence of both of the parental strains. Our experiments were results with WEHI-3 CSA, NZB marrow cells gave at least not designed to discern small sex-related differences, but at least normal numbers of colonies in cultures stimulated by LCCM one of each type of analysis was done with males and females, or ES CSA, and the plateau of colony numbers normally seen and it seemed that the sex of the animals did not appreciably with high CSA concentrations was not observed (Fig. 4). influence the results. Prostaglandin Sensitivity of Colony Formation. Previous

300f . 104 0

U U c g 250 0 IA0 4) E 'a i E 200 c 0 I 150 0 0 . *

cD ; VZ 100IF 0 d 3 103:7 S ._a C 50 / d 0 /0 o d1 102- U 1:64 1:16 1:4 un-1:64 1:16 1:4 un- dil. dii. CSA dilution 5 10 15 20 25 30 35 40 45 Age, weeks FIG. 4. Differential stimulation by various granulocyte-macrophage CSA sources. Dilutions of semipurified WEHI-3-M (v), LCCM FIG. 3. Incidence in spleen of NZB mice of B cells that form (-), or endotoxin-stimulated serum (ES) (0) CSA prepared as de- colonies in LPS-potentiated cultures containing 25 pAg of purified scribed in Materials and Methods were added to cultures of NZB anti-p antibodies per ml. (Left) or CBA (Right) bone marrow cells. Downloaded by guest on September 28, 2021 Immunology: Kincade et al. Proc. Natl. Acad. Sci. USA 76 (1979) 3467

2 CSA alone CSA + 0.1 AM PGE u 250 250

0L- E 200 200

6, I~~~~~~I C 0 *a * 0~ .0 150 -o 150 S~~~~~~~~

* 0 o 100 100 -A . ~ : 'aG 0 ~~~~~~~~~~~-T *'D 50 I - 50 0 -06 10-8 10-6 0 C. _1 PGE, M i4+

FIG. 5. Sensitivity of granulocyte-macrophage colony formation of CBA/H @) and NZB (I) cells to PGE. In this experiment CBA/H

+ -- and NZB bone marrow formed 150 5 and 55 i 3 colonies per 105 cultured cells, respectively, in unfractionated WEHI-3-M stimulated cultures (Left) containing no prostaglandin. In LCCM-stimulated FIG. 7. Incidence of cultures (Right) the uninhibited control values were 88+i5 for CBA/H granulocyte-macrophage progenitors and and 185 i 3 for NZB cells. their sensitivity to prostaglandin in parental and hybrid progeny mice. Assays were done with the same individuals shown in Fig. 6 and unfractionated WEHI-3-M as the source of CSA. DISCUSSION

The two salient findings of this study were: (i) an exceptional present in partially immunodeficient CBA/N mice (13). The type of B cell, which proliferates in LPS-potentiated semisolid minimal culture requirements for colony formation are 2- cultures and resists anti-g antibodies, exists in NZB lymphoid mercaptoethanol, fetal calf serum, and appropriate mitogens tissues, and (ii) defective responsiveness of hemopoietic pre- such as those native to laboratory grade agar (12). Modification cursor cell populations to certain types of colony-stimulating of these basic conditions, such as the use of macrophage feeder activity was demonstrated. These abnormalities were partially layers, addition of LPS, or addition of SRBC are done to opti- expressed in F1 hybrids with two different strains and thus may mize colony size and numbers and, most importantly, to achieve be under codominant genetic control. consistent, linear dose-response curves (14). Indirect observa- The B cells that proliferate to form colonies in agar cultures tions suggest that the populations of B cells that respond under have been extensively characterized. Clonable cells are almost these different conditions may not be completely overlapping. as heterogeneous as all other B cells in terms of physical prop- More colonies develop in cultures in which more than one erties and other respects, and the only population of slg+ cells potentiator is used, and occasionally this number equals the sum obtained with each one used alone (12, 14). Adult bone marrow that have been found to totally lack cloning potential are those B cells have a higher cloning efficiency in LPS- than in SRBC-potentiated cultures (15, 25). NZB mice develop very large numbers of cells that clone in LPS-containing cultures, C whereas the incidence of clonable cells in SRBC-containing a,a 106 cultures was usually similar to that of normal CBA/H mice. - . Others have found that B cells in very young NZB mice are () polyclonally activated to differentiate and secrete immunoglobulin, and the autoimmune potential has been transferred c to irradiated normal recipients with cells other than T lym- .r, phocytes (3, 7, 8). These findings suggest that abnormal B-cell .sE 10 0 0 regulatory mechanisms could be important in the pathogenesis of this disease. We found that clonable B cells from NZB mice c were normally susceptible to inhibition by PGE and anti-6 0 antibodies. In the latter case, nanogram quantities of io4104 purified antibodies achieved a plateau of inhibition, and in other studies we determined 4-1 that the anti-6 resistant B cells include some cells that are surface 6- and others that are 6+ (unpublished results). Our results thus do not contradict other reports that NZB B cells have a low density of cell surface IgD relative to IgM (20), but indicate that the 6 receptors on their clonable cells function normally in regulation. As predicted from all of our previous studies, the B cells of NZB mice do not clone in anti-cs containing, SRBC-potentiated cultures. However, a substantial 4-§ q- K- C, proportion of the LPS-stimulated clones resist anti-1 suppres- 11,v~o sion. These cells were completely sensitive to inhibition by polyvalent anti-Ig and thus are B cells, and, because they were FIG. 6. Heritability of anti-j resistant B colony-forming cells in depleted by incubation in anti-g or anti-IgM coated petri dishes, F1 hybrid offspring of NZB mice. Individual values together with they may actually express sIgM. The maximum incidence of means for each group are given for 17-week-old NZB, 18-week-old these unusual B cells was found in adult NZB lymph nodes, and NZBxNZW, 18-week-old CBAxNZB, 10-week-old NZW, and 9- this would indicate that they belong to relatively mature and week-old CBA/H mice. perhaps memory cell populations. Downloaded by guest on September 28, 2021 3468 Immunology: Kincade et al. Proc. Nati. Acad. Sci. USA 76 (1979)

Our fortuitous observation that NZB and A/J mice differ in 1. Krakauer, R. S., Waldman, T. A. & Strober, W. (1976). J. Exp. IgD allotypes is of interest with respect to immunoglobulin Med. 144, 662-673. genetics. These mice share other Ig allotypes, and recombina- 2. Cantor, H., McVay-Boudreau, L., Hugenberger, J., Naidorf, K., tion of Ig allotype genes is an extremely rare event in the mouse Shen, F. W. & Gershon, R. K. (1978) J. Exp. Med. 147, 1116- (21). The Ig-5 (IgD) locus could be distal to the other Ig allotype 1125. genes but must nonetheless be linked closely to them because 3. DeHeer, D. & Edgington, T. S. (1978) J. Immunol. 118, NZB and C57BL/6.Ige mice share all allotype alleles (22, 1858-1863. 23). 4. McCombs, C., Hom, J., Talal, N. & Mischell, R. I. (1975) J. Im- Where lymphocytes require mitogens for clonal prolifera- munol. 115, 1695-1699. tion, precursors of eosinophilic, megakaryocytic, erythroid, 5. Warner, N. L. & Moore, M. A. S. (1971) J. Exp. Med. 134, granulocytic, and monocyte-macrophage cells require factors 313-334. collectively referred to as CSA for colony formation in vitro (26, 6. Datta, S. K., Manny, N., Andrzejewski, C., Andre-Schwartz, J. 27). A wide variety of normal and neoplastic cells elaborate & Schwartz, R. S. (1978) J. Exp. Med. 147,854-871. CSA, and the diversity of these substances, their nature, and the 7. Moutsopoulos, H. M., Boehm-Truitt, M., Kassan, S. S. & Chused, relationship between these different molecules is far from un- T. M. (1977) J. Immunol. 119, 1639-1644. 8. Izui, S., McConahey, P. J. & Dixon, F. J. (1978)J. Immunol. 121, derstood. Myeloid progenitors in NZB marrow were almost 2213-2219. totally refractory to a CSA moiety in semipurified WEHI-3-M 9. Klassen, L. W., Lynell, W., Krakauer, R. S. & Steinberg, A. D. over a wide concentration range. In contrast, at least normal (1977) J. Immunol. 119,830-837. numbers of colonies were obtained with LCCM or ES CSA, and 10. Shirai, T., Hayakawa, K., Okumura, K. & Tada, T. (1978) J. it is interesting that numbers of NZB colonies did not plateau Immunol. 120, 1924-1929. with high concentrations of these stimulators. This perhaps 11. Morton, J. I. & Siegel, B. V. (1974) Proc. Nati. Acad. Sd. USA 71, explains the elevated numbers of colonies previously observed 2162-2165. with NZB cultures containing ES (28). In addition to these ab- 12. Kincade, P. W., Ralph, P. & Moore, M. A. S. (1976) J. Exp. Med. normalities, the sensitivity of NZB precursors to PGE was dif- 143, 1265-1270. ferent from those in normal CBA marrow. The different CSA 13. Kincade, P. W. (1977) J. Exp. Med. 145,249-263. are known to differ in terms of the morphology of the colonies 14. Kurland, J. I., Kincade, P. W. & Moore, M. A. S. (1977) J. Exp. they elicit, but this does not correspond to the NZB abnormality. Med. 146, 1420-1435. Most preparations of semipurified WEHI-3-M preferentially 15. Kincade, P. W., Paige, C. J., Parkhouse, R. M. E. & Lee, G. (1978) stimulate neutrophil colonies and LCCM elicits almost pure J. Immunol. 120, 1289-1296. 16. Williams, N., Eger, R. R., Moore, M. A. S. & Mendelsohn, N. macrophage colonies (16, 29). However, NZB precursors made (1978) Differentiation 11,59-3. at least normal numbers of granulocyte colonies in ES and failed 17. Pearson, T., Galfre, G., Ziegler, A. & Milstein, C. (1977) Eur. J. to respond to batches of WEHI-3-M that were significantly Immunol. 7,684-690. contaminated with monocyte-macrophage stimulators. Further 18. Oi, V. T., Jones, P. P., Goding, J. W., Herzenberg, L. A. & study is required to determine whether one or more subsets of Herzenberg, L. A. (1978) in Lymphocyte Hybridoma8, eds. myeloid precursors is absent in NZB marrow or whether these Melchers, F., Warner, N. L. & Potter, M. (Springer, Basel, Swit- cells are defective in their recognition of certain regulators. We zerland). have also found (unpublished data) that (i) NZB marrow cells 19. Wysocki, L. J. & Sato, V. L. (1978) Proc. Natl. Acad. Sci. USA survive and function poorly under continuous marrow culture 75,2844-2848. conditions that permit proliferation and differentiation of cells 20. Cohen, P., Ziff, M. & Vitetta, E. S. (1978) J. Immunol. 121, of other mouse strains for many weeks, (ii) abnormalities in in 973-977. vitro myelopoiesis are apparent with early fetal liver cells and 21. Herzenberg, L. A. & Herzenberg, L. A. (1978) in Handbook of can be transferred to lethally irradiated BDF1 recipients, (iii) Experimental Immunology, ed. Weir, D. M. (Blackwell, Oxford), NZB precursors 3rd Ed., pp. 12.1-12.23. are abnormal in responsiveness to certain other 22. Coding, J. W., Warr, G. W. & Warner, N. L. (1976) Proc. Nati. regulatory substances, and (iv) these phenomena are probably Acad. Sci. USA 73,1305-1309. due to intrinsic cellular abnormalities rather than active sup- 23. Goding, J. W., Scott, D. W. & Layton, J. E. (1977) Immunol. Rev. pressor mechanisms. 37, 152-186. None of the unusual features that we describe here for NZB 24. Kurland, J. & Moore, M. A. S. (1977) Exp. Hematol. 5, 357- mice are necessarily involved in their autoimmune disease. 373. Further study is also required to determine if and how poly- 25. Lala, P. K., Johnson, G. R', Battye, F. L. & Nossal, G. J. V. (1979) clonal B cell activation events may be related to nonlymphoid J. Immunol. 122,334-341. abnormalities. In the meantime NZB mice may prove useful 26. Metcalf, D. (1977) In Vitro Cloning of Normal and Leukemic for dissecting hemopoietic precursor cell populations and the Cells (Springer, New York). macromolecules that regulate their function. 27. Burgess, A. W., Metcalf, D. & Russell, S. (1978) in Differentiation of Normal and Neoplastic Hematopoietic Cells, Cold Spring We are grateful to Drs. J. Goding and N. Warner for providing en- Harbor Conferences on Cell Proliferation, eds. Clarkson, B., couragement, advice, reagents, and mice. Dr. P. Ralph maintained the Marks, P. A. & Till, J. E..(Cold Spring Harbor Laboratory, Cold hybridoma cell lines and made helpful comments on the manuscript. Spring Harbor, NY), Vol. 5, Book A, pp. 339-357. We thank Mr. R. R. Eger for providing unique preparations of semi- 28. Metcalf, D. & Stevens, S. (1972) Cell Tissue Kinet. 5, 433- purified GSA. This work was supported by Grants AI-12741, AI-11843, 446. CA-17404, CA-08748, AG-00541, and Research Career Development 29. Stanley, E. R. & Guilbert, L. J. (1979) in Mononuclear Phago- Award AI-00265 from the U.S. Public Health Service, and by the cytes-Functional Aspects, ed. van Furth, R. (Nijhoff, The National Foundation-March of Dimes. Hague, Netherlands), in press. Downloaded by guest on September 28, 2021