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(Journal of Leukocyte Biology. 2000;68:144-150.)
© 2000 by Society for Leukocyte Biology

The colony-stimulating factors and collagen-induced arthritis: exacerbation of disease by M-CSF and G-CSF and requirement for endogenous M-CSF

Ian K. Campbell, Melissa J. Rich, Robert J. Bischof and John A. Hamilton

Inflammation Research Centre, The University of Melbourne, Department of Medicine, Royal Melbourne Hospital, Parkville, Victoria 3050, Australia


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
There is increasing evidence that the colony-stimulating factors (CSFs) may play a part in chronic inflammatory autoimmune diseases, such as rheumatoid arthritis (RA). We examined the involvement of macrophage CSF (M-CSF or CSF-1) and granulocyte CSF (G-CSF) in collagen-induced arthritis (CIA), a murine model of RA. Daily injections of M-CSF or G-CSF, 20–24 days postprimary immunization with type II collagen, exacerbated disease symptoms in suboptimally immunized DBA/1 mice. Support for the involvement of endogenous M-CSF in CIA was obtained by studies in which neutralizing monoclonal antibody reduced the severity of established CIA and also by studies showing the resistance of M-CSF-deficient op/op mice to CIA induction. These studies show that M-CSF and G-CSF can be proinflammatory in CIA and provide evidence that macrophage- and granulocyte-lineage cells can exacerbate CIA. Our results also show that M-CSF-dependent cells are essential for CIA development, suggesting M-CSF may be a suitable target for therapeutic intervention in RA.

Key Words: rheumatoid arthritis • hematopoiesis • cytokines • inflammatory mediators • in vivo animal models


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The colony-stimulating factors (CSFs) are a family of four cytokine growth factors originally identified by their ability to support the proliferation and differentiation of haemopoietic progenitor stem cells into mature monocytes/macrophages and granulocytes [for review, see ref. 1 ]. The CSF family comprises the lineage-specific members, macrophage CSF (M-CSF or CSF-1) and granulocyte CSF (G-CSF), and the nonlineage-specific members, granulocyte-macrophage CSF (GM-CSF) and multi-CSF [also called interleukin (IL)-3]. Although known for their actions in haemopoiesis, the CSFs in vitro also exhibit certain activities that could predispose them toward a proinflammatory role in vivo [1 , 2 ].

There is increasing evidence that CSFs may play a major part in chronic inflammatory autoimmune diseases, such as rheumatoid arthritis (RA). Three of the four known CSFs (M-CSF, G-CSF, and GM-CSF) are produced by human-joint tissue cells (chondrocytes, synovial fibroblasts) in vitro in response to the inflammatory cytokines, IL-1 and tumor necrosis factor {alpha} (TNF-{alpha}) [3 4 5 6 ]. Certain CSFs have also been identified in RA joint exudates [7 , 8 ] and in RA synovial tissues by in situ hybridization [9 ]. We recently obtained more direct evidence of a role for CSFs in inflammatory arthritis by demonstrating that GM-CSF can exacerbate disease symptoms when injected into mice sensitized to develop collagen-induced arthritis (CIA) [10 ] and that GM-CSF-deficient mice are resistant to the induction of CIA [11 ]. Because GM-CSF can affect cells of the monocytic, granulocytic, and dendritic cell lineages, its key target cell in autoimmune arthritis remains obscure.

The ability of M-CSF and G-CSF to affect specific haemopoietic cell lineages presents a unique opportunity to dissect and assess the relative contributions of two major cellular pathways of GM-CSF action in CIA and to gain a better understanding of their separate contributions to disease. Although a previous study [12 ] described exacerbation of disease in the Streptococcus agalactiae rat model of arthritis by intravenous (i.v.) injection of recombinant human (rh) M-CSF, little is known of the actions of this cytokine in an autoimmune model of arthritis, such as CIA, or whether endogenous M-CSF is required for disease development. Also, to our knowledge, there is only one study describing the effect of G-CSF on an animal arthritis model [13 ]. This is surprising given that it has been administered to patients with Felty’s syndrome (a variant of RA) for treatment of neutropenia [14 15 16 17 18 ] and is being used for the mobilization of haemopoietic stem cells in patients with severe RA prior to myeloablation [19 ].

The aim of this study was to obtain a better understanding of the role of M-CSF and G-CSF in autoimmune arthritis. Several approaches were used to investigate the involvement of M-CSF, and to a lesser extent G-CSF, in CIA. We show that exogenous M-CSF and G-CSF can exacerbate arthritis symptoms when injected into mice suboptimally primed to develop CIA. We also show that endogenous M-CSF is required for CIA development, because disease severity is reduced in arthritic mice treated with neutralizing monoclonal antibodies (mAbs) to M-CSF, and CIA could not be established in M-CSF-deficient (op/op) mice. These findings extend the proinflammatory potential of the CSF family. Our results also suggest possible new targets for therapeutic intervention in RA and suggest caution should be exercised when using G-CSF for therapeutic purposes in RA.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents
Chick type-II collagen (CII) and incomplete Freund’s adjuvant were purchased from Sigma Chemical Co. (St. Louis, MO); heat-killed Mycobacterium tuberculosis (H37Ra) and lipopolysaccharide (LPS) (Eschericia coli strain 0111:B4) were purchased from Difco Laboratories (Detroit, MI). rhM-CSF (specific activity 8x107 units/mg by M-NFS-60 cell proliferation assay) was a gift from Chiron Corp. (Emeryville, CA); rhG-CSF and rhIL-1ß (specific activity 5x108 units/mg; units as defined in ref. 3 ) were obtained from Amgen Inc. (Thousand Oaks, CA). Rat mAb to M-CSF, 5A1, and its isotype control mAb, DX48 [rat immunoglobulin G1 (IgG1) raised to digoxin], were gifts from F. Kull (Glaxo-Wellcome, Research Triangle Park, NC). The neutralizing capacity of 5A1 was 7 x 105 units of M-CSF per mg 5A1. M-CSF and G-CSF were carrier-free and were diluted in phosphate-buffered saline (PBS) or normal saline for experimentation.

Mice
Male DBA/1 mice (7–11 weeks old) were purchased from the Animal Resources Centre (Canning Vale, Western Australia) and allowed to acclimate for 1 week before experimentation. DBA/1 mice were fed standard rodent chow and water ad libitum and were housed in sawdust-lined cages in groups of five. Osteopetrotic (op/op) mutant mice and their littermate wild-type controls, each on the C57BL/6 and C3HeB/FeJ background, were obtained from the Ludwig Institute for Cancer Research (Parkville, Victoria, Australia) and maintained in micro-isolators within the Ludwig Institute’s animal house facility. Following weaning, the op/op mice, which lacked incisors, were maintained on a diet containing equal amounts of powdered chow and Ensure nutritional powder (VHA Trading Co., Mulgrave, Victoria, Australia), mixed in sterile water.

Collagen-induced arthritis
An emulsion was formed by dissolving 2 mg/ml chick CII overnight at 4°C in 10 mM acetic acid and combining it with an equal volume of complete Freund’s adjuvant (CFA), prepared by adding heat-killed M. tuberculosis to incomplete Freund’s adjuvant at 5 mg/ml. All mice were injected intradermally (i.d.) at several sites into the base of the tail with a total of 100 µl emulsion containing 100 µg CII and 250 µg M. tuberculosis. Depending on the experiment, mice were then treated in one of three ways.

(1) Exacerbation studies (suboptimum CIA)
Mice in this group received no further injections of CII. This procedure followed that previously described [10 ], where omission of a booster injection resulted in suboptimal induction of CIA. After different periods of time, mice were given 4–5 daily injections of M-CSF or G-CSF, or the same volume of vehicle at the doses, routes, and times indicated in the text.

(2) Neutralizing mAb studies (optimum CIA)
In experiments to examine the effect of neutralizing mAbs, mice were immunized for maximum CIA response by administration of a repeat of the primary CII injection as a boost at day 21, as previously described [11 ]. Mice were then assessed daily for clinical signs of CIA and, from 27 days postprimary immunization onward, newly arthritic mice were randomly assigned to one of two mAb treatment groups—5A1 or DX48. The arthritic mice were then given 10 consecutive daily intraperitoneal (i.p.) injections of mAb (300 µg), and the arthritis was assessed clinically throughout.

(3) IL-1 enhancement of CIA (optimum CIA)
In some experiments (indicated in text), the rapid onset of CIA was induced, as described [20 ], by four consecutive daily subcutaneous (s.c.) injections into the rear footpad with IL-1ß (20 ng, 104 units). The injections were initiated at 21 days postprimary CII immunization, and no CII boost was given.

Clinical and histological assessment of arthritis
Mouse limbs were assessed for redness and swelling, and a clinical score was allocated 2–3 times per week for up to 60 days. The scoring system was as previously described [10 ], where 0 = normal, 1 = slight swelling, 2 = extensive swelling, and 3 = joint distortion and/or rigidity. The maximum score per mouse was 12. When killed, the limbs of the mice were removed, fixed, decalcified, and processed for histological assessment of haematoxylin and eosin-stained sections, as previously described [11 ].

Detection of Abs to CII by enzyme-linked immunosorbent assay (ELISA)
ELISAs for Abs to CII were performed as previously described [11 ]. Horseradish peroxidase-conjugated goat anti-mouse IgG (Sigma) detection Ab was used. Standard curves were constructed from sera of CII hyperimmunized DBA/1 mice (arbitrarily set at 1000 units/ml).

Statistics
The following statistical tests were used to compare different groups for the parameters indicated: Mann-Whitney two-sample rank test (clinical scores), Student’s t-test for the difference of two means (anti-CII Ab levels), and the {chi}2 test (arthritis incidence). For each test, P < 0.05 was considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Exogenous M-CSF exacerbates CIA
We previously showed that rhGM-CSF exacerbates CIA in suboptimally immunized DBA/1 mice when injected at days 21–24 postprimary CII immunization [10 ]. Because GM-CSF can affect the function of cells in the macrophage and granulocyte lineages [1 ] and is also implicated in dendritic cell development [21 ], it was, therefore, of interest to examine the effects of the lineage-specific CSFs, namely M-CSF and G-CSF, in this model. Table 1 summarizes data from four experiments, where DBA/1 mice were suboptimally immunized by i.d. injection of chick CII in CFA, without boost, and then injected with different doses of M-CSF via the routes indicated. The M-CSF doses used were based on a previous study [22 ] in which a 10-fold increase in circulating mature monocytes was obtained in mice given four daily injections of 20 µg rhM-CSF. The time of administration was according to our previous studies showing GM-CSF exacerbation of CIA [10 ] and is considered to be at a stage when the mice have an established immune response to CII but prior to CIA development [23 ].


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  		Table 1. Effect of M-CSF on Murine CIAa

 
There was a general trend of increased disease severity (clinical score) in the M-CSF-treated mice for all four experiments (Table 1) . Statistical significance was obtained in two experiments (Experiments 1 and 3), where M-CSF potently exacerbated the CIA response in terms of increased disease incidence and severity. In three other experiments (unpublished results), M-CSF had no effect; in these experiments, the control groups generally showed a higher basal level of disease (Table 1 , and unpublished results). The variable response to M-CSF was independent of the injection regime employed; histological analyses reflected the clinical assessments (unpublished results). When data from all seven experiments were combined, there were significantly more CIA-positive mice in the M-CSF-treated groups (39/69) than in the control groups (26/68) by day 40 (P<0.05, {chi}2 analysis). The variation observed is unlikely to be a result of endotoxin contamination in the M-CSF, because this was < 0.1 ng/mg M-CSF (limulus lysate assay). Furthermore, no effect of LPS was observed, alone or with M-CSF, when CII-immunized mice were given four daily s.c. injections (days 20–23) of M-CSF (15 µg), or vehicle, with and without the addition of 100 ng LPS (unpublished results).

The kinetics of the enhanced CIA response to M-CSF is shown for Experiment 3 in Figure 1 , as incidence (A) and clinical score (B). In that experiment, mice were given four consecutive daily i.p. injections of M-CSF, beginning 20 days postimmunization with CII. It is evident in these suboptimally immunized mice that, although M-CSF generated an earlier and more severe form of CIA compared with the control mice, its effect was not immediate but delayed, with no notable response occurring until five days after the initial M-CSF injection. The mean clinical scores peaked at day 43.



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  		Figure 1. Kinetics of the effect of exogenous M-CSF on CIA incidence and severity. DBA/1 mice were immunized by i.d. injection of CII in CFA and 20 days later, were given four consecutive daily i.p. injections of M-CSF (30 µg) or the same volume of vehicle (n=10 mice per group). Mice were assessed and scored for clinical signs of arthritis, as described in Materials and Methods. The maximum clinical score per mouse was 12. Results show the percent cumulative incidence (A) and mean ± SEM clinical scores (B) of the two groups of mice up to day 60. The average clinical scores of the M-CSF-treated mice were significantly greater than for the vehicle-treated mice over the 21–60-day period (P<0.005).

 
Requirement for endogenous M-CSF in CIA
The above data showed that in some experiments M-CSF could exacerbate suboptimum CIA. By analogy with our previous studies in which GM-CSF exacerbated suboptimum CIA [10 ], and GM-CSF deficiency prevented disease [11 ], it was reasoned that endogenous M-CSF may also play a role in conventional CIA. To address this, the effect of neutralizing mAb to M-CSF on established CIA was investigated. This time, DBA/1 mice were immunized for optimum induction of CIA by primary and boost injections with CII in CFA, as previously described [11 ], and given 10 daily injections of neutralizing mAb to M-CSF (5A1) or isotype-control mAb (DX48), beginning the first day of onset of arthritis for each individual mouse. Figure 2 shows that anti-M-CSF mAb alleviated the clinical severity of CIA established in mice after day 27. Similar results were obtained in a second experiment.



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  		Figure 2. Neutralizing mAbs to M-CSF reduce the severity of established CIA. DBA/1 mice were immunized by i.d. injection of CII in CFA at days 0 and 21. Mice were clinically assessed daily for signs of arthritis, and from day 27 onward, newly arthritic animals (shown as day 1 of arthritis) were assigned to one of two mAb-treatment groups—anti-M-CSF (5A1) or isotype control (DX48). Each group received 10 consecutive daily i.p. injections of mAb (300 µg per day), beginning the first day of arthritis symptoms. Results show the mean ± SEM (n=10 mice per group) clinical scores of the two groups of mice over the first 11 days of CIA. The anti-M-CSF-treated group had significantly lower clinical scores than the control group over the 4–7-day period (P<0.05).

 
To further assess the role of endogenous M-CSF in CIA, M-CSF-deficient mice (op/op) and their wild-type controls were compared for their responses in the CIA model. Our previous studies [11 ] have shown that, with appropriate protocols, CIA of high incidence and severity can be induced in mice of non-DBA/1 background, including the background strain of the op/op mouse (i.e., C57BL/6xC3HeB/FeJ) [24 ]. Therefore, op/op and wild-type control mice were immunized by i.d. injection with a single dose of CII in CFA, and optimum CIA onset was accelerated by daily injection (days 21–24) into the rear footpad with IL-1ß, as previously described for DBA/1 mice [20 ]. IL-1ß caused only mild transient swelling in the injected paws of the op/op mice, which was completely resolved by day 29. In contrast, the wild-type mice exhibited significantly more severe arthritis (increased paw swelling and clinical score, see Table 2 ), which persisted beyond day 29 (five of eight mice), progressing to joint stiffening and the involvement of other noninjected paws. In conclusion, the op/op mice were relatively resistant to the induction of arthritis by IL-1 enhancement of CIA, consistent with the neutralizing mAb studies (above) indicating a requirement for M-CSF in CIA development.


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  		Table 2. M-CSF-Deficient (op/op) Mice Are Resistant to CIA Inductiona

 
Exogenous G-CSF exacerbates CIA
The effect on CIA of administration of the other lineage-specific CSF, G-CSF, was also examined. Suboptimally stimulated DBA/1 mice were given four consecutive daily injections (days 18–23) with different doses of G-CSF (Table 3 ). The effect of G-CSF was dose-dependent (see Experiment 1, Table 3 ), and in contrast to M-CSF, G-CSF (30 µg) consistently elicited a strong response in each of three experiments. When data from the three experiments were combined, there were significantly more CIA-positive mice at day 38 in the G-CSF-treated groups (30 µg) (27/30) compared with the control groups (15/30; P=0.001, {chi}2 analysis). Therefore, G-CSF increased the incidence and severity of CIA. Figure 3 shows the kinetics of the G-CSF effect on CIA incidence (A) and severity (B) (Experiment 3 from Table 3 ). Again, in contrast to M-CSF, the effect of G-CSF was rapid (within 2 days of the first G-CSF injection), and the mean clinical score peaked earlier (day 27).


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  		Table 3. G-CSF Exacerbates Murine CIAa

 


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  		Figure 3. Kinetics of the effect of exogenous G-CSF on CIA incidence and severity. DBA/1 mice were immunized by i.d. injection of CII in CFA and 20 days later, were given four consecutive daily i.p. injections of G-CSF (30 µg) or the same volume of vehicle (n=10 mice per group). Mice were assessed and scored for clinical signs of arthritis, as described in Materials and Methods. Results show the percent cumulative incidence (A) and mean ± SEM clinical scores (B) of the two groups of mice up to day 60. The average clinical scores of the G-CSF-treated mice were significantly greater than for the vehicle-treated mice over the 21–60-day period (P<0.005).

 
Humoral response to CII in M-CSF- and G-CSF-treated mice
The humoral response to CII is an important component of CIA development in mice [24 25 26 ]. The anti-CII IgG sera levels of mice treated with M-CSF (Experiment 3 from Table 1 ) or G-CSF (Experiment 3 from Table 3 ) were, therefore, examined to determine whether these correlated with the increased CIA incidence and severity observed in CSF-treated mice compared with controls. Figure 4 shows that the anti-CII IgG levels of the M-CSF-treated, but not the G-CSF-treated, mice were higher at day 26 and day 60 timepoints, compared with the corresponding controls.



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  		Figure 4. Effect of M-CSF and G-CSF on serum anti-CII IgG levels. In two separate experiments (A and B), DBA/1 mice were immunized by i.d. injection of CII in CFA, as described in Materials and Methods, without boost. At days 20–23, mice (n=10) were injected daily i.p. with 30 µg of M-CSF or G-CSF, or with vehicle (control). Sera were prepared from blood collected at 26 and 60 days postimmunization and assayed for anti-CII IgG levels by ELISA. *P < 0.05 compared with vehicle control.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The CSFs are recognized as regulators of haemopoietic cell proliferation, differentiation, and survival and more recently as potent activators of mature haemopoietic cell function [1 , 2 ]. Our previous studies have shown that the nonlineage-specific CSF, GM-CSF, can exacerbate CIA [10 ] and that GM-CSF-deficient mice are protected from developing CIA [11 ], suggesting a role for this cytokine in inflammatory autoimmune arthritis. Because GM-CSF can affect cells of the myeloid and granulocyte lineages [1 ], and each is thought to be involved in CIA [27 ], we examined the effects of the lineage-specific CSFs, M-CSF and G-CSF, in this model. We found that M-CSF and G-CSF can similarly exacerbate suboptimum CIA. Interestingly, the effect of G-CSF on CIA was more immediate and consistent than that of M-CSF (cf. Figs. 1 and 3 ). The ability of myeloid and granulocyte lineage-specific CSFs to exacerbate CIA suggests that the potent effect of GM-CSF previously demonstrated on CIA [10 ] could mean that it involves the coordinated actions of more than one cell lineage. This study provides further support for the idea that the CSFs can have a proinflammatory function and so may be suitable targets for therapeutic intervention in RA.

The requirement for endogenous M-CSF for CIA was demonstrated by a reduction in the severity of established disease by M-CSF-neutralizing mAb (Fig. 2) and by the resistance of M-CSF-deficient (op/op) mice to arthritis development (Table 2) . M-CSF could be functioning in CIA through effects on monocyte-macrophage infiltration into the joints, on type-A synovial lining (macrophage lineage) cells, or on osteoclasts [28 ]. Because administration of rhM-CSF at the doses used in this study elicits up to a 10-fold increase in circulating blood monocyte levels in mice [22 ], this, in turn, could result in increased numbers of monocytes/macrophages entering the joint. The importance of monocyte/macrophage infiltrate in CIA has been demonstrated by the requirement for macrophage migration inhibitory factor (MIF) function in the model [29 ] and by the prevention of adoptive transfer of CIA by Mac-1 blockade [30 ]. Enhanced numbers of activated macrophages are present in the synovium at the earliest preclinical stages of CIA [27 ] and are an important source of cytokines for disease development [31 , 32 ]. In addition, macrophages may function as antigen-presenting cells for CII [33 ].

The absence of CIA in op/op mice (Table 2) possibly provides support for the importance of the type-A synovial lining cells in this model. Although op/op mice have normal T-cell-dependent immune responses [34 ], they lack type-A synovial cells [28 ]; thus, the latter are considered an M-CSF-dependent cell population. Interestingly, unlike some macrophage populations, type-A synovial cell deficiency in op/op mice cannot be overcome by the systemic injection of M-CSF from birth [28 ]. These cells may, therefore, be dependent on local rather than systemic M-CSF production for their survival and development. This could be supplied by the fibroblast-like (type B) synovial cells or chondrocytes, which produce basal levels of M-CSF at least in vitro [4 , 6 , 35 ]. Our findings with the op/op mouse are in agreement with previous studies in which depletion of type-A cells from the synovial lining by injection of liposome-encapsulated clodronate resulted in protection from CIA [36 ]. However, the inability of circulating rhM-CSF to restore type-A synovial cells in the op/op mouse [28 ] suggests that the effect of exogenous M-CSF on CIA could be at the level of circulating monocytes/macrophages (see above). In short, the numbers and function of resident and infiltrating monocyte-macrophage populations could be affected by M-CSF in CIA [27 ]. Finally, M-CSF is essential for osteoclast production [37 ] and function [28 ], and these cells could be contributing to the bone destruction observed in the late stages of CIA. The osteopetrosis of the op/op mouse is a direct result of the absence of osteoclasts in these mice.

A threshold anti-CII IgG response is considered a necessary but alone insufficient requirement for CIA development [25 , 26 , 38 ]. The anti-CII IgG levels of the M-CSF-treated mice were higher than those of control mice (Fig. 4A) , but whether this increase actually contributed toward the exacerbation of CIA by M-CSF is uncertain. The effect of M-CSF on anti-CII IgG levels could be through elevated levels of cytokines, such as IL-6, possibly because of increased numbers of "primed" monocytes/macrophages capable of producing them [39 40 41 ]. In keeping with previous studies in the op/op mouse [34 ], these mice developed normal levels of anti-CII IgG (unpublished results), similar to our observations with the GM-CSF-deficient mouse, which showed resistance to CIA but a normal humoral response to CII [11 ]. The inability of exogenous G-CSF to modulate anti-CII IgG levels (Fig. 4B) yet still exacerbate CIA (Table 3 , Experiment 3) suggests that the threshold Ab level for subsequent disease development was already achieved, even in these suboptimally immunized mice. Therefore, G-CSF would appear to enhance disease through a separate process. Further experimentation is required to clarify the mechanisms involved, but these observations concur with other studies [42 ] in which it was shown that anti-CII Ab levels do not necessarily correlate with disease.

Data from various animal models suggest that M-CSF may be a key mediator of certain autoimmune diseases. The MRL/lpr mouse has elevated M-CSF serum levels, which precede spontaneous glomerulonephritis [43 ]. Splenic mononuclear phagocytes of the autoimmune motheaten mouse spontaneously produce M-CSF [44 ] and have an enhanced proliferative capacity in vitro [45 ]. In a rat model of experimental allergic encephalomyelitis (EAE), spinal chord extracts showed elevated mRNA expression for M-CSF and c-fms (M-CSF receptor), which correlated with the clinical incidence of disease [46 ]. The present study extends this list to autoimmune arthritis. Perhaps in support of our findings, a recent gene-mapping study in B10.RIII x RIIIS/J mice identified a non-MHC CIA susceptibility gene (Mcia2) on chromosome 3 in the region of the M-CSF locus—a region previously linked to EAE [47 ].

This study has also provided evidence that cells of the granulocytic lineage, specifically neutrophils, are important in CIA. Only one study has described the effect of G-CSF in an arthritis model [13 ], where it was found that i.v. injection of rhG-CSF into rats enhanced the passive transfer of arthritis using suboptimal doses of anti-CII rat sera with an associated induction of peripheral blood neutrophilia. We have now shown an effect of G-CSF on actively immunized murine CIA. The effect of G-CSF in the current study could be a result of an increased number of circulating neutrophils mobilized from haemopoietic stem cells, an enhanced functional ability of the neutrophils, and/or enhanced haemopoietic progenitor stem-cell numbers in the circulation. In vivo administration of G-CSF is known to cause rapid systemic neutrophilia and haemopoietic stem-cell mobilization [48 ]. G-CSF, when given at the elicitation but not the priming phase, enhances delayed-type hypersensitivity possibly through an effect on mononuclear cell recruitment [49 ]. Because mononuclear cells lack G-CSF receptors, this recruitment occurs indirectly through the granulocytes, possibly via chemokine production. A similar mechanism could be operating in the present study. Thus, in addition to a direct effect on granulocyte levels/activation, there may be an indirect effect on mononuclear cell recruitment leading to accelerated CIA in the already-CII-primed mice. As well as monocytes/macrophages and neutrophils, GM-CSF stimulates the proliferation of eosinophils [1 ], and so the latter cells could also be contributing to the exacerbation of CIA in our studies with GM-CSF [10 ]. However, the absence of receptors for G-CSF and M-CSF on eosinophils precludes a direct effect on these granulocytes in the present study.

Several studies have demonstrated arthritis flare in RA patients following administration of G-CSF for correction of the neutropenia occurring as a complication of Felty’s syndrome [15 16 17 18 ] or as a consequence of drug treatments [50 ]. In view of this, two recent studies have addressed the safety of G-CSF administration to RA patients for haemopoietic stem-cell mobilization and subsequent autologous stem-cell transplantation. In one study [51 ], no arthritis flare was noted, although only low G-CSF doses were used (5 µg/kg), and all five patients had received corticosteroid treatment before therapy. A second study described arthritis flare in two of five patients receiving 10 µg/kg G-CSF, the optimum dose of two doses tested for satisfactory stem-cell mobilization [52 ]. Even higher doses of G-CSF may be required to adequately mobilize haemopoietic stem cells in RA patients. We showed that G-CSF doses as low as 0.3 µg per mouse (10 µg/kg) had detectable effects on CIA severity (Table 3 , Experiment 1), observations consistent with the latter study. Our findings further highlight the need for caution in administering G-CSF to arthritis patients, particularly if doses beyond 10 µg/kg are to be considered. Additional studies are required to determine whether endogenous G-CSF is necessary for CIA development. An approach using neutralizing Abs to G-CSF and/or G-CSF gene-knockout mice similar to that described herein for M-CSF and elsewhere for GM-CSF [11 ] would address this.

In summary, we have shown that the lineage-specific CSFs, M-CSF, and G-CSF can exacerbate CIA, findings supporting roles for monocytes/macrophages and granulocytes in autoimmune inflammatory arthritis. We further showed that M-CSF blockade can alleviate disease and that M-CSF-dependent cells are required for CIA. The presence of CSFs in rheumatoid joints and their mutual modulation in vitro by the proinflammatory cytokines, IL-1 and TNF-{alpha}, led to the proposal of a local "CSF network" within joints that might help explain the chronicity of inflammatory joint disease [53 ]. Together with our previous findings [10 , 11 ], the present study highlights the potent proinflammatory potential of the CSFs and suggests they may be targets for therapeutic intervention in rheumatoid disease.


   ACKNOWLEDGEMENTS
 
This work was supported by grants from the NHMRC of Australia and in part by grants from the Arthritis Foundation of Australia and Amgen. We thank J. Davis for animal care, Dr. A. Dunn (Ludwig Institute for Cancer Research) for the supply and maintenance of op/op and littermate mice, and Dr. F. Kull (Glaxo-Wellcome) for supply of 5A1 and DX48 mAbs.


   FOOTNOTES
 
Correspondence at current address: Dr. Ian K. Campbell, Reid Rheumatology Laboratory, Division of Autoimmunity and Transplantation, The Walter and Eliza Hall Institute of Medical Research, Post Office Royal Melbourne Hospital, Victoria, 3050, Australia. E-mail: campbell{at}wehi.edu.au

Received November 29, 1999; revised February 29, 2000; accepted March 1, 2000.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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