CLINICAL

IMMUNOLOGY

AND

IMMUNOPATHOLOGY

Vol. 65, No. 3, December, pp. 212-218, 1992

Expanded Macrophage Precursor Populations in BXSB Mice: Possible Reason for the Increasing Monocytosis in Male Mice M. R. HADAM,*

G. VIETEN,~ Department

of Immunology,

Department

of Internal

H. DE BOER, A. OLP, M. FRICKE, AND K. HARTUNG Medicine, and *Department Hannover, Germany

The BXSB mousespontaneously developsan autoimmune diseasethat resembleshuman systemic lupus erythematosus (SLE). During their lifetime, male BXSB mice show an increasing monocytosis in the peripheral blood as opposedto their female littermates. This monocytosis is unique among autoimmune-prone mice. To test the hypothesis that alterations at the stem cell level may be responsible for this monocytosis, myeloid bone marrow precursor cells were examined in both male and female BXSB mice from 4 to 40 weeks of age. The number of M-CSF responding stem cells (CFU-M) and the number of GM-CSF responding stem cells (CFU-GM) were higher than in all other inbred mouse strains tested. In addition, male BXSB mice developed a progressive increase of CFU-M and CFU-GM in the bone marrow during their lifetime, which paralleled the peripheral blood monocytosis. The monocytosis in male BXSB mice is the result of a further expansion of the strainspecific high number of macrophage precursors by intrinsic factors, which may be attributed to the influence of the Yaa factor. The sex-specific expanded mononuclear phagocyte system may promote the autoimmune process and may be one reason for the dramatic course of murine SLE in male BXSB mice. 0 1992 Academic Press, Inc. INTRODUCTION

The BXSB mouse strain was established in 1987 by Murphy and Roths after mating a C57BW6J female with a SB/Le male (1, 2). BXSB mice spontaneously develop a progressive and lethal autoimmune disease which is regarded as an experimental mouse model for human systemic lupus erythematosus (SLE) (3). These mice manifest clinical and immunological abnormalities, such as moderate lymphnode enlargement, splenomegaly, impaired T cell function, B cell hyperactivity seen as hypergammaglobulinemia, spontaneous polyclonal antibody production, and secretion of various autoantibodies, which leads to immune complex deposition in the kidney (4-8). 1 To whom reprint requests should be addressed at Abt. Immunologie und Transfusionsmedizin, Medizinische Hochschule Hannover, Konstanty-Gutschow-Str. 8, 3000 Hannover 61, Germany. 212 0090-1229/92 Copyright All rights

$4.00 0 1992 by Academic Press, of reproduction in any form

Inc.

reserved

of Pediatric

Surgery,

Hannover

Medical

School.

The manifestation of these autoimmune phenomena is remarkably accelerated in male BXSB mice, which have a 50% mortality at 5 months compared to 15 months for female mice. This is attributed to the effect of an unmapped single gene, designated as Yaa gene (Y-chromosome-linked autoimmune accelerator), which is located on the Y chromosome. As Miyawaki and colleagues demonstrated by genetic studies, the Yaa gene can hardly induce autoimmune disease in non-autoimmune strains, but it is able to accelerate the development of autoimmune disease in strains with a genetic predisposition for autoimmunity (g-12). By the investigation of the cellular basis of BXSB male-dominant disease, using male to female or female to male bone marrow transfers, or by marrow transplantation across major histocompatibility complex barriers, it was shown that the pace of BXSB disease is determined entirely by the donor stem cells and not at all by the environment in which these cells develop (13, 14). Unlike human lupus and other experimental models of autoimmunity, BXSB male mice develop an increasing monocytosis in the peripheral blood during their lifetime (15). This phenomenon has so far only been described by Wofsy in 1984, who characterized the cells as Thy l-, Lyt l-, Lyt 2-, ThB-, Ig-, Nk 1.1-, Ia-, and T200+, and Mac-l + cells. The etiology and the possibly pathophysiological significance of this monocytosis have not been clarified yet. The monocytosis in male BXSB mice may be of relevance for the disease because alterations of macrophage functions and monokine production have been reported in human and murine SLE (16-19). These findings may be important because macrophages can influence B and T cell activity. Our studies were performed to investigate the mononuclear phagocyte system (MPS) of the BXSB mouse. In this paper alterations in the bone marrow (BM) stem cell population of BXSB mice are reported. The findings indicate a close relationship between the change at the stem cell level and the monocytosis in the peripheral blood of male BXSB mice.

MACROPHAGE MATERIALS

AND

PRECURSOR POPULATIONS

METHODS

Mice. BXSB mice were obtained from The Jackson Laboratories (Bar Harbor, ME) and an own breeding colony was established behind barriers at the Central Animal Facility of the Medical School of Hannover. The animals were regularly controlled and free of pathogens as listed in GV-SOLAS, 1988 (20). C57BL/ 6J, NMRI, DBA/W, and NZB/W mice were kindly provided by the Central Animal Facility and by Dr. A. Emmendorffer, Fraunhofer Institute, Hannover. BALB/c mice were purchased from the Central Institute for Laboratory Animal Breeding, Hannover, and SB/Le mice from The Jackson Laboratories. Cell preparation. Peripheral blood mononuclear cells (PBMC) were obtained by retroorbital bleeding into heparinized pipettes. Mononuclear cells were isolated by separation over Ficoll (Biochrom KG, Berlin, Germany) and washed twice in RPM1 1640 (Flow Laboratories GmbH, Meckenheim, Germany). BM was obtained from the femurs of mice by rinsing with a 25gauge needle. Mononuclear cells (BMMC) were separated on Ficoll gradient and, after two washes with Iscoves modified Dulbecco’s medium (IMDM; Biochrom KG), the cells were used in the stem cell assays. Colony stimulating factors (CSF). CSFs were obtained as follows: murine recombinant IL-3 and murine recombinant GM-CSF (both from Genzyme, Boston MA), human recombinant M-CSF (a kind gift of Dr. D. Krumwieh, Behringwerke, Marburg, Germany), and a supernatant of the fibroblast cell line L929 (L929 Sup.), containing M-CSF activity (a kind gift of Professor Dr. M.-L. Lohmann-Matthes, Fraunhofer-Institut, Hannover, Germany). If not indicated otherwise the following final concentrations were used for stimulation: IL-3,62.5 U/ml; GM-CSF, 125 U/ml; M-CSF, 125 U/ml; L929Sup., 100 l&ml of 1:3 diluted stock solution.

213

IN BXSB MICE

mixed granulocyte/macrophage colonies (24). Eosinophi1 colonies were detected atter staining with solvent blue 38 (Sigma, Deisenhofen, Germany). All cultures were performed in three to four independent experiments, each done in triplicates. The mean and standard deviations were calculated. FACS analysis. PBMC were analyzed with a fluorescence-activated cell sorter (Becton-Dickinson 440) according to methods previously described (25). The following monoclonal antibodies were used for the analysis of subpopulations: Mac-l (TIB 128, ATCC), Thy 1.2, Lyt-1, Lyt-2, and L3T4 (all from BectonDickinson GmbH, Heidelberg, Germany). Statistics. Statistical differences were calculated employing the Wilcoxon rank-sum test, and P values below 0.05 were considered as significant. RESULTS

Stimulation with M-CSF. In the initial experiments BMMC from BXSB mice as well as control mice were cultured for 7 days in the presence of ret M-CSF, or in the presence of L929 Sup., as a crude source of M-CSF, in different concentrations. As shown in Fig. 1 BMMC of 12-week-old BXSB male mice produced seven times more macrophage colonies than age-matched C57BL/6J control mice after maximal stimulation with 125 U/ml ret M-CSF. With higher M-CSF dilutions (15.6 U/ml; 7.8 U/ml> no significant differences between BXSB and C57BL/6J mice were observed. This indicates that the CFU-M of the BXSB mice do not respond more sensitively to ret M-CSF than control mice. The same results were found in a dose-response curve with L929 Sup. But in contrast to the stimulation with ret M-CSF male and female BXSB mice showed the same number of precursor cells after stimulation with L929 Sup. BMMC from BXSB mice and C57BL/6J mice were BXSBmale o BXSB female

l

The stem cell assays were performed as essentially described (21-23). Briefly, BMMC were plated at 1 x lo5 cells/ml basic medium in 35-mm petri dishes (Tecnomara, Fernwald, Germany) in the presence of L929 Sup, ret M-CSF, GM-CSF, or IL-3 at the concentrations indicated. The basic medium consisted of IMDM containing 100 III/ml penicillin, 100 pg/ml streptomycin, 2 mM L-glutamine, 15% fetal calf serum (FCS; GIBCO, Eggenstein, Germany), and 0.35% agar (Difco, Detroit, MI). The cultures were incubated in a humidified atmosphere with 5% CO, at 37°C. On Day 7 colonies (~50 cells) were counted. The colonies were then transferred onto slides, dried, and finally stained with naphthol-AS-nchloroacetate (Sigma, Deisenhofen, Germany) and ol-naphthyl butyrate (Merck, Darmstadt, Germany) to identify granulocyte, monocyte/macrophage, and Bone marrow stem cells assay.

units hu ret M-CSF 1. Stimulation with human ret M-CSF dilutions. The colony growth (CFU-M) of 1 x 10’ BMMC from la-week-old male BXSB mice (filled circle), female BXSB mice (open circle), and control C57BI&J mice (diamond) is shown. Results are expressed as mean colony counts (*SD) of four experiments, each performed in triplicate. FIG.

214

VIETEN

then analyzed at 8, 20, and 40 weeks. Figure 2 shows the result after stimulation with 125 U/ml ret M-CSF. C57BLEJ control mice produced an average of 140 colonies at all ages. BXSB male mice, however, produced 192 colonies at the age of 8 weeks, At 20 weeks of age the number of colonies (289) was significantly higher than in agematched C57BLEJ mice. Up to 450 colonies were produced by BMMC of 40-week-old male BXSB mice. These findings show that there is a progressive increase of CFU-M in the BM during the lifetime of male BXSB mice. Female BXSB mice produced more colonies than C57BLEJ mice, but the difference was not significant, and the number of colonies remained constant through the examination period of 40 weeks (Fig. 2). The differences in male and female colony numbers were also found after 5 and 14 days of cultivation. Initially performed continuous observation could exclude differences in growth kinetics of male and female colonies and clusters. After stimulation with L929 Sup. the results differed somewhat from those that were obtained with ret M-CSF. The number of colonies in both male and female BXSB mice was significantly higher than the number of colonies in C57BW6J control mice (Fig. 3). At no time was there a significant difference in the colony number between male and female BXSB mice. During the test period the number of macrophage colonies remained constant at a high level (>200 colonies) in both sexes of BXSB mice (Fig. 3). We then tested a number of different mouse strains and found that all strains including the lupus-prone NZBiW mouse and the diabetic C57BL/KS dbl + +/m mouse produced lower numbers of colonies than the CFU-M 400

(rec.

M-CSF

stimulation)

1

ET AL. CFU-M 400

20 in weeks

FIG. 2. Colony growth after stimulation with 125 U/ml hum. rec. M-CSF, CFU-M of 1 x 10’ BMMC from 8-,20-, and 40-week-old male BXSB mice (black columns), female BXSB mice (hatched columns), and control C57BLW mice (dotted columns) are compared. Results are expressed as mean values (?SEM) of three experiments, each performed in triplicate. (*P < 0.05)

Sup.

stimulation)

-

B age

20 in weeks

40

FIG. 3. Colony growth after stimulation with L929 Sup. The I 1 10s BMMC were stimulated with 100 al of the 1:3 diluted stock solution. Colony formation of 3, 20-, and 40-week-old male BXSB mice (black columns), female BXSB mice (hatched columns), and control C57BL/6J mice (dotted columns) are compared. Results are expressed as mean values (tSEM1 of four experiments, each performed in triplicate.

BXSB mice (Fig. 4). This also applies to the SBiLe mouse, one of the gene sources of the BXSB strain (1). In all cultures tested with M-CSF (recombinant or L929 Sup.), only pure macrophage/monocyte colonies were produced (>95%), as ascertained by esterase staining. This is in accordance with the physiological function of M-CSF which is primarily a stimulus for monocytes/macrophages. Stimulation with GM-CSF. The results, which compare the stimulation with ret GM-CSF of male and female BXSB mice and C57BLEJ mice, are summarized in Fig. 5. In young animals there were no significant differences in the number of GM-CSF reactive precursor cells. At 20 weeks of age the colony number of male BXSB mice was significantly higher than in control C57BL/6J mice. Whereas the number of colo-

C57BLIKS C57BL/KS C57BL/KS

age

CL929

BALE/c CBA CD DBA NMRI ob/ob db/+ +/m l /+ C57BLIBJ SBlLe

BXSB BXSB

m f L

0

50

1

100 number

I

150 of colonies

200

250

FIG. 4. Colony growth (CFU-M) from 1 x lo5 BMMC of different mouse strains. Cells were stimulated with L929 Sup. Mean values (2SEM) of three independent experiments are shown.

MACROPHAGE

PRECURSOR POPULATIONS

c FU-GEMM

CFU-GM 12Or

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IN BXSB MICE

100

‘r 80 80 80 80 40 40 20

20

0 8 age

20 in weeks

40

0 8 age

20 in weeks

40

FIG. 5. Colony growth after stimulation with 125 U/ml mur. rec. GM-CSF, CFU-GM of 1 x lo5 BMMC from 8-, 20-, and 40-week-old male BXSB mice (black columns), female BXSB mice (hatched columns), and control C57BLK.I mice (dotted columns) are compared. Results are expressed as mean values (-+SEM) of three experiments performed in triplicate. (*P < 0.05)

FIG. 6. Colony growth after stimulation with 62.5 U/ml murine rec. IL-3. CFU-GEMM of 1 x lo6 BMMC from 8-, 20-, and 40-weekold male BXSB mice (black columns), female BXSB mice (hatched columns), and control C57BWJ mice (dotted columns) are compared. Results are expressed as mean values (*SEMI of three experiments performed in triplicate.

nies remained constant in female BXSB mice until 40 weeks of age (mean colony number of 631, it increased continuously in male BXSB mice. At the age of 40 weeks male BXSB mice reached an average number of 90 colonies (range between 61 and 111 colonies), in contrast to 37 colonies in control C57BL/6J mice. Morphologic analysis of the colonies showed granulocyte, mixed granulocyte-macrophage, and monocytemacrophage colonies, which did not differ significantly in proportions between the groups of mice tested.

mals. Ten to 15% of the PBMC were positive for Mac-l. Most of these cells showed the typical morphology of monocytes: indented nuclei and coarse chromatin (microscopic analyses). Beginning with 12 weeks of age, we found a progressive increase of monocytes in male BXSB mice. At the age of 20 weeks 50% of the PBMC were Mac-l + and the monocytosis reached an average of 74% (range 60 to 88%) in 40-week-old BXSB males. The percentage of Mac-l + cells in the PBMC of C57BLN mice remained constant during the experimental period of 40 weeks, whereas a slight increase was observed in older female BXSB mice (up to 37%). For further characterization of the blood leucocytes, we performed functional tests with the same cell populations, as tested in the FACS. We found that PBMC of old male BXSB mice produced large amounts of PGE, and oxygen-radicals (0,-j. The amounts of these mediators correlated well with the increase of Mac-l + cells (manuscript in preparation). Female BXSB mice and C57BL/6J healthy control mice produced only low amounts of PGE, and oxygen radicals. These findings and the experiments performed by Wofsy (15) support the assumption that these Mac-l + cells are monocytic cells.

Stimulation with IL-3. The response of BMMC to IL-3 was similar in all three groups tested (Fig. 6). At 8 weeks of age a mean number of 30 colonies was observed in all strains. At the age of 20 weeks the number of IL-3 responding precursor cells increased slightly, the number of colonies amounting up to 42 colonies in C57BL&I, 55 colonies in female BXSB mice and 63 colonies in male BXSB mice. At 40 weeks of age the number of precursor cells remained constant in C57BL/6J mice and female BXSB mice. Male BXSB mice showed an increase in the number of colonies, but the difference was not significant between the groups of mice tested. As previously observed after stimulation with GM-CSF there was no significant difference in the colony morphology. In all cultures the percentage of eosinophilic colonies was sl%. Flow cytometric analysis ofPBMC. PBMC from male and female BXSB mice, and C57BW6J mice were analyzed at different ages (6,12,16,20, and 40 weeks) using monoclonal antibodies to quantify monocytes, T and B cells. A representative survey of the alteration of the Mac1 + subpopulation is displayed in Fig. 7. In all three groups tested there were no differences in young ani-

DISCUSSION

A large body of evidence has shown that the autoimmune disease of BXSB mice is determined in the hematopoietic stem cell population. In 1980 Eisenberg et al. (14) could show by stem cell transplantation that the course of the autoimmune disease is determined entirely by male BM or spleen cells. Ikehara et al. (26) reported that allogeneic bone marrow transplantation (ABMT) ameliorates established nephritis in BXSB mice, and long-term observation (from 5 months to 1 year post-ABMT) revealed that the autoimmune dis-

VIETEN

BXSB male

BXSB female

16 weeks

Log10 Fluorescence

Intensity

(3 Decades)

FIG. 7. Indirect staining of PBMCs from male BXSB mice (A, C, E) and female BXSB mice (B, D, F) with Mac-l. Animals were tested at 6,16, and 40 weeks of age. Data are presented as flow histogramm (one representative experiment out of nine similar experiments).

eases of BXSB and NZB/W mice often remained corrected for at least 1 year after ABMT. Also a preprogramming of autoimmunity at the stem cell level is known for the other autoimmune mouse strains (NZB/W, MRL lprllpr) (2730). Our studies were undertaken to investigate whether an alteration at the BM stem cell level may be the cause for the monocytosis in male BXSB mice. We could show that BXSB mice possess a considerably higher number of strain-specific myeloid precursor cells than all other mouse strains tested, including the autoimmune NZB/W strain. The fact that the increase of macrophage precursors takes place in both sexes, as was shown in particular by stimulation with L929 Sup., indicates that the expansion of the monocytic lineage is a racial characteristic, probably caused by a genetically determined high number of myeloid precursor cells. Although both male and female BXSB mice have high numbers of precursor cells already in early life, only male BXSB mice develop a monocytosis in the peripheral blood, and the autoimmune disease is more prominent in male mice. We could show that this monocytosis is preprogrammed by an increase in CFUGM and CFU-M in the BM of male BXSB mice. In seeking an interpretation for the different observation in the response to L929 Sup., a crude source of M-CSF and to ret M-CSF it must be kept in mind that unfractionated L929 supernatant contains some other stimulating factors in addition to M-CSF (31-34). Further, it is known that there is an additive or a synergistic effect if M-CSF is used in combination with other growth factors (35). We therefore suggest that BXSB

ET AL.

mice possess a strain-specific, genetically determined high number of a subpopulation of macrophage precursor cells which possess in addition to the M-CSF receptors receptors for other growth factors. To gain their full proliferative potential, these cells require the combination of several stimulating factors. This hypothesis is supported by the results of Breen et al. (36), who found that there is an additive effect of the combination of GM-CSF, M-CSF, and IFN-y on the proliferation of murine macrophage progenitor cells. Only male BXSB mice are enabled to proliferate and differentiate (possibly caused by the influence of the sex-specific Yaa factor). Therefore male mice develop a progressive increase of CFU-GM and CFU-M after stimulation with rec. CSF. One possible reason for the sex-specific increase of CFU-GM and CFU-M in male BXSB mice could be a dysregulation of the hematopoietic system caused by an altered response to inhibitory factors. To examine this hypothesis, we investigated the reactivity of CFU-M to inhibitory factors like PGE,, IFN-11. and TNF-a. We observed no differences in the inhibitory effects of these substances in different concentrations on the CFU-M of BXSB mice as compared to healthy C57BL6J mice (data not shown). Our findings that (1) CFU did not proliferate spontaneously in BXSB mice and (2) no different response to inhibitory factors could be observed indicated that one or more external regulating factors may stimulate the proliferation of macrophage precursors in male BXSB mice. This lead to the assumption that there is a specific upregulation of the MPS in male BXSB mice. We have detected colony-forming capacity in the plasma of male BXSB mice. All colonies were identified as pure macrophage colonies. No stimulating capacity was observed in serum of female BXSB mice or C57BLi 6J mice (own unpublished results). Miiller et al. (37~ found similar results with serum from MRL lprilpr mice, and Yui et al. (38) reported that MRL Zprllpr mice have an increased level of circulating M-CSF detectable as early as 1 week of age. It has further been shown that M-CSF is increased in the circulation of mice with bacterial infections (39) and in the synovial fluid of patients with rheumatoid arthritis (40). It may therefore well be that in conjunction with the strain-specific high number of macrophage precursors, M-CSF alone or in cooperation with other factors, may lead to the monocytosis in BXSB mice. Since the Yaa factor is known to accelerate the autoimmune process, the unusual occurrence of the stimulating factors may be caused by this sex-specific factor. Further experiments are needed to clarify the correlation between the Yaa factor and the activation of the MPS. One can speculate that the activation of the MPS is an important factor in the development of murine lupus because (1) in male BXSB mice the monocytosis

MACROPHAGE

PRECURSOR

appears very early in life and (2) Ikehara et al. (41) have mentioned incidentally that after ABMT the number of peripheral blood monocytes will return to normal values. Other clues are given by Miiller et al. (37), who have shown that already very early in life the macrophage system is strongly expanded in BM, spleen, and liver of MRL Zprllpr mice. They examined the organ-associated macrophage system of two autoimmune mouse strains (NZB/W and MRL Zprllpr) and could demonstrate that in these strains the number of both macrophages and macrophage precursors in liver and spleen is considerably increased. The fact that hematopoietic activity in these two lupus-prone mouse strains is not down-regulated during the first weeks of life suggests a causative role of macrophages in the autoimmune process. We have also found high numbers of CFU-M in spleen and liver of young and old male BXSB mice (manuscript in preparation). This suggests that a hyperproliferative macrophage system may be important not only for the inflammatory process in affected organs, as has been shown for lupus nephritis by Boswell and Kelley (42, 43), but also for the induction of the autoimmune disease process. This assumption is supported by experimental results of Lee and colleagues (44), who achieved an improvement of autoimmune diabetes by macrophage depletion in BB rats, and by Kolb and Kolb-Bachofen (45) who found that macrophages are the earliest infiltrating cells in the pancreas of autoimmune diabetic BB rats. Considering these results, the dramatical course of the disease in male BXSB mice (50% mortility at the age of 5 months) is caused by the influence of the expanded and activated MPS. Our final conclusions are: (1) BXSB mice possess a strain-specific genetically determined high number of macrophage precursors; (2) in male mice this population is further expanded by intrinsic factors, which may be attributed to the influence of the Yaa-factor; and (3) an activation of the MPS may be an essential condition for the development of the murine SLE. Further experiments are required to determine the causative part of the macrophage system in lupus in general and in BXSB-mice in particular. ACKNOWLEDGMENTS This work was supported by research Grant 01 VM 8606 from the Bundesministerium fiir Forschung und Technologie (BMFT), Germany. We are indebted to Dr. D. A. Monner and to Professor Dr. K. Giirtner for helpful suggestions. REFERENCES 1. Murphy, E. D., and Roths, J. B., New inbred strains. Mouse News Lett. 58, 51-52,1978. 2. Murphy, E. D., and Roths, J. B., Autoimmunity and lymphoproliferation: Induction by mutant gene lpr, and acceleration by a male-associated factor in strain BXSB mice. In “Genetic Control

POPULATIONS

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