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Receptor-Deficient Mice Is Conditioned by Mycobacteria in Freund’s Adjuvant and by Increased Expansion of Mac-1+ Myeloid Cells1
*
Rega Institute, University of Leuven, Leuven, Belgium;
Children’s Hospital, Harvard Medical School, Boston, MA 02115; and
Laboratory of Histochemistry, Faculty of Medicine, University of Leuven, Leuven, Belgium
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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acts as a
disease-limiting factor in the pathogenesis of the disease. Here we
show that in collagen-induced arthritis the protective effect of
IFN- depends on the presence of mycobacteria in the adjuvant.
Omission of mycobacteria inverts the role of endogenous IFN- to a
disease-promoting factor. Thus, the mycobacterial component of CFA
opens a pathway by which endogenous IFN- exerts a protective effect
that supersedes its otherwise disease-promoting effect. Extramedullary
hemopoiesis and expansion of the Mac-1+ cell population
accompanied the accelerated and more severe disease course in the
IFN- receptor knockout mice immunized with CFA. Treatment of such
mice with Abs against the myelopoietic cytokines IL-6 or IL-12
inhibited both disease development and the expansion of the
Mac-1+ population. We postulate that mycobacteria in CFA
stimulate the expansion of the Mac-1+ cell population by a
hemopoietic process that is restrained by endogenous IFN-. These
results have important implications for the validity of animal models
of autoimmunity to study the pathogenesis and to evaluate
cytokine-based therapy of autoimmune diseases. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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in the pathogenesis of experimental models
of autoimmune diseases has been the subject of several studies (for
review, see Ref. 1). A frequently used approach has
consisted of examining changes in disease, incidence, and severity
following ablation of IFN- by administration of neutralizing
anti-IFN- Abs or by gene knockout technology. In some models,
the outcome of such studies has been indicative of a disease-promoting
role of IFN-. Examples are spontaneous lupus nephritis in (NZB
x NZW)F1 mice (2, 3, 4) or in
MRL-lpr mice (5), diabetes in nonobese diabetic
mice (6, 7, 8), and orchitis following autoimmunization
without adjuvant (9). Well-documented cellular and
molecular pathways exist by which IFN- can exert this
disease-promoting effect, including potentiation of autoantigen
presentation by enhanced expression of the MHC, activation of
macrophages to produce inflammatory mediators, and orientation of Th
cell differentiation in the Th1 rather than the Th2 direction.
In several autoimmune disease models, the outcome of IFN- ablation
procedures has been supportive of a protective rather than a
disease-promoting role. Such examples are experimental autoimmune
encephalomyelitis (10, 11, 12, 13, 14, 15, 16), experimental autoimmune
uveitis (17, 18), and collagen-induced arthritis
(CIA)3 (19, 20). So
far, a satisfactory explanation, based on established cellular and
biological effects of IFN- has not been available for this
protective effect. The protective effect of IFN- in experimentally
induced encephalomyelitis also contrasts with apparent
disease-promoting role in transgenic mice overexpressing IFN-
(21). It has been proposed that IFN- produced locally
in affected tissues might indeed be disease-promoting, but that this
effect might be overruled by systemically produced IFN- acting as a
protective factor by its antiproliferative action on certain
mononuclear cell populations or by its ability to induce suppressor
cells.
We initiated the present study as we noted that all three models in
which endogenous IFN- exerts a distinct protective role, rely on the
use of the heat-killed mycobacteria-containing adjuvant, CFA, for
immunization of the animals. IFN- is well known to be essential for
effective host defense against mycobacteria, so that the use of these
organisms as adjuvant can be surmised to add a level of complexity that
might explain the protective effect. For our analysis of the
interaction between heat-killed mycobacteria and IFN-, we chose CIA
as a model rather than experimental autoimmune encephalomyelitis,
because we found that CIA can be readily induced, albeit with less
efficiency, using IFA, which does not contain mycobacteria. The outcome
of our analysis is that endogenous IFN- acts as a disease booster of
CIA induced by an IFA-assisted autoimmunization protocol, but protects
against enhanced CIA induced by the classical CFA-assisted
autoimmunization schedule. In addition, we obtained evidence for CIA
pathogenesis to be controlled by a Mac-1+
population whose expansion is enhanced by mycobacteria but curbed by
IFN-.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The generation and the basic characteristics of the mutant mouse
strain (129/Sv/Ev) with a disruption in the gene coding for the
-chain of the IFN- receptor (IFN-R KO) have been described
(22). These IFN-R KO mice were back-crossed with DBA/1
wild-type mice for 10 generations to obtain the DBA/1 IFN-R KO mice
used in the present study. IFN-R KO and wild-type mice were bred in
our Experimental Animal Centre of the University of Leuven. The
experiments were performed in 8- to 12-wk-old mice, but in each
experiment, the mutant and wild-type mice were age-matched with a 5-day
limit. The male to female ratio was kept between 0.8 and 1.3 in each
experimental group.
Induction of CIA and clinical assessment of arthritis
Native chicken collagen type II (CII; Elastin Products Company, Owensville, MO) was dissolved in 0.05 M acetic acid at 2 mg/ml by stirring overnight at 6°C and emulsified in an equal volume of IFA or CFA containing 1.5 mg/ml heat-killed Mycobacterium butyricum (Difco, Detroit, MI). Mice were sensitized with a single 100-µl intradermal injection of the emulsion at the base of the tail. Mice were examined daily for signs of arthritis. The disease severity was recorded following a scoring system for each limb: 0, normal; 1, redness and/or swelling in one joint; 2, redness and/or swelling in more than one joint; 3, redness and/or swelling in the entire paw; 4, deformity and/or ankylosis.
Histological examination
Spleens and fore and hind limbs were fixed in B5. Limbs were subsequently decalcified overnight with formic acid. Four-micron-thick paraffin sections were stained with hematoxylin and eosin. Severity of arthritis was evaluated using three parameters: infiltration of mono- and polymorphonuclear cells, hyperplasia of the synovium, and pannus formation. Each parameter was scored on a scale from 0 to 3 (absent, weak, moderate, and severe).
In vivo Ab treatments
Monoclonal Abs were produced from hybridomas grown by i.p.
inoculation in Pristane-primed athymic nude mice (nu/nu of nuclear
magnetic resonance imaging background). Neutralizing mAb against murine
IFN- (F3, rat IgG2a) (10) was
purified by affinity chromatography on a mouse anti-rat 
-chain
mAb. The neutralizing titer (end-point dilution corresponding to 50%
neutralization of the antiviral effect of 30 U/ml of mouse IFN- on
mouse L929 cells challenged with mengovirus) was
105.3 U/ml (IgG content, 1.4 mg/ml). A
neutralizing rat IgG2a Ab against murine IL-12 was produced using
hybridoma C17.8 (kindly provided by Dr. G. Trinchieri, Wistar
Institute, Philadelphia, PA). The Ab was purified by affinity
chromatography on protein G (Pharmacia, Uppsala, Sweden). Ab against
murine IL-6 was prepared from ascites fluid from thymusless nude mice
inoculated with the 20F3 (rat x mouse) hybridoma (American Type
Culture Collection, Manassas, VA). This rat IgG1 Ab was purified by
affinity chromatography on an anti-rat -chain Ab-Sepharose
column. The neutralizing titer (endpoint dilution corresponding to 50%
neutralization of the cell growth effect of 10 U of murine IL-6 per ml)
was 105.5 (IgG content, 2.9 mg/ml). Irrelevant
rat IgG2a was used as an isotype control and was prepared from ascites
fluid of a rat plasmocytoma obtained through the courtesy of Dr. H.
Bazin (University of Louvain Medical School, Brussels, Belgium). The
IgG was purified by anion exchange chromatography on Hiload Q Sepharose
and gel filtration on Superdex 200 (Pharmacia). Batches of
anti-IFN-, anti-IL-12, anti-IL-6, and irrelevant IgG2a
were tested for endotoxin content by a chromogenic Limulus
amoebocyte lysate assay (KabiVitrum, Stockholm, Sweden) and were found
to contain <2 ng/ml endotoxin. Injections were given in 200 µl of
pyrogen-free saline.
Serum anti-CII Ab levels
Blood samples were taken from the orbital sinus and were allowed
to clot for 1 h at room temperature and at 4°C overnight.
Individual sera were tested for Abs directed to chicken CII by ELISA as
described (19). A serial 2-fold dilution series of a
purified standard preparation was included in each experiment to allow
for calculation of the Ab content. The standard material was purified
by affinity chromatography from pooled sera obtained from several
IFN-R KO and wild-type arthritic mice and contained 0.5 mg/ml
IgG.
Delayed-type hypersensitivity (DTH) reactivity
At the indicated days, symptom-free mice were challenged in the right footpad with 10 µg chicken CII in 20 µl PBS. The left footpad received 20 µl PBS. DTH response was measured as percent swelling (i.e., the difference between the thickness of the right and left footpads, as percent of the thickness of the left footpad) at 24 and 48 h postchallenge.
Characterization of spleen cells
Spleens were gently cut into small pieces and passed through cell strainers (Becton Dickinson Labware, Franklin Lakes, NJ). RBC were lysed by two consecutive incubations (5 and 3 min, 37°C) of the suspension in NH4Cl (0.83% in 0.01 M Tris/HCL, pH 7.2). Remaining cells were washed, resuspended in cold PBS, and counted. Aliquots of 2 x 105 cells in 0.2 ml were preincubated (30 min) with an Fc receptor-blocking Ab (2.4G2; PharMingen, San Diego, CA; 1 µg/ml) and then stained for 30 min with PE-conjugated anti-Mac-1 Ab (CD11b+; PharMingen). PE-conjugated rat IgG (clone A 95-1; PharMingen) was used as an isotype-identical control. Cells were analyzed by a FACScan flow cytometer (Becton Dickinson, San Jose, CA). Differential leukocyte counts were obtained by centrifuging (700 rpm for 8 min) aliquots of 2 x 105 cells onto slides (Cytospin apparatus, Shandon, Cheshire, U.K.) and staining with Hemacolor (Merck, Darmstadt, Germany). From each spleen cell suspension, at least 200 cells were counted.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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R KO mutation results in higher susceptibility for CIA
induced by CFA-assisted immunization, but protection against CIA
induced by IFA-assisted immunization
Groups of IFN-R KO or wild-type mice were immunized with
CII in either IFA or CFA. The animals were scored daily for
clinical signs of arthritis. The time of disease onset, cumulative
incidences, and maximal clinical scores recorded in a total of 7
experiments are summarized in Table I
.
The data confirmed the well-known lower incidence and the delayed onset
of arthritis in wild-type mice immunized with Ag in IFA instead of CFA:
only 25% of the mice developed overt arthritis when immunized using
IFA, against 63% using CFA. In line with our previously reported
findings (19), IFN-R KO mice proved to be more
susceptible than wild types for induction of CIA elicited with CII
in CFA, as was evident from their significantly earlier times of
disease onset and increased cumulative incidence and disease
scores.
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R KO mice showed clinical signs of arthritis,
whereas such signs did develop in 25% of the wild-type mice given the
same immunization schedule. Histological examination of limbs collected
on day 64 confirmed development of classical arthritic lesions in
wild-type but not in IFN-R KO mice (Fig. 1): infiltration of the synovium by mono-
and polymorphonuclear cells, synovial hyperplasia, and pannus
formation. Results of quantitative scoring of histological sections are
shown in Fig. 2. All histological
parameters of arthritis were lower in mutant than in wild-type mice.
Furthermore, synovial hyperplasia and pannus formation were evident in
limbs of clinically symptomless wild-type mice. The proportions of
inflamed limbs with histological scores 
2 for infiltration,
hyperplasia, and pannus were 0, 0, and 8% in IFN-R KO mice
(n = 12) vs 28, 56, and 61% in wild-type mice
(n = 18) and 0, 38, and 46% in symptom-free wild-type
mice (n = 13).
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R KO mice given the IFA-assisted
immunization schedule is associated with reduced Ab and DTH responses
to CII
Fig. 3A describes the
results of two experiments in which anti-CII Ab was determined
in blood samples taken at different time points during the course of
arthritis development in IFN-R KO and wild-type mice given the
IFA-assisted immunization schedule. Significantly lower levels of
anti-CII IgG were observed in IFN-R KO than in wild-type
mice. All isotypes (IgG1, IgG2a, and IgG2b) were similarly affected
(data not shown).
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B). Significant swelling was
observed in wild-type but not in IFN-R KO mice, indicating that DTH
reactivity was reduced in the mutant mice. We also examined the
possibility that the Th1/Th2 cytokine balance evolved differently in
IFN-R KO than in wild-type mice, as previously found to be the case
when CFA is used (19). However, following exactly the same
assessment system (measurement of IL-2 or IL-4 serum levels after an in
vivo anti-CD3 challenge), we found the Th1/Th2 balance of IFN-R
KO mice immunized with the IFA-assisted schedule not to be different
from that of similarly immunized wild-type mice (data not shown).
Endogenous IFN- prevents the expansion of a splenic
Mac-1+ cell population, elicited by the mycobacterial
component of CFA
Our results indicate that the mycobacterial component of CFA
triggers events that not only enhance and accelerate pathogenesis of
CIA, but in addition open a pathway by which endogenous IFN- can
exert a protective effect that supersedes its natural disease-promoting
effect. A possible clue to this mycobacterium-dependent pathway was the
observation of a pronounced splenomegaly occurring in the IFN-R KO
mice immunized with CII in CFA. For instance, in one experiment in
which the mice were sacrificed on day 23 postimmunization, the mean
spleen weights (±SEM, n = 4) of IFN-R KO mice and
wild-type mice was, respectively, 226 ± 18 and 125 ± 12 mg
(CFA-assisted immunization); 106 ± 7 and 93 ± 6 mg
(IFA-assisted immunization); and 77 ± 14 and 70 ± 5 (naive
mice). To clarify the possible significance of this splenomegaly for
the disease course, spleen sections were examined histologically and
spleen cell suspensions were analyzed morphologically and by FACScan
using sets of Abs for different leukocytes: T lymphocytes
(CD3+, CD4+, and
CD8+), NK cells (IL-2Rß+,
CD3-, and CD8-), NKT
cells (IL-2Rß+, CD3+, and
CD8-), B lymphocytes
(CD19+ and B220+), and
monocytes/macrophages/granulocytes (CD11ß+ or
Mac-1+). Histological examination performed on
day 24 postimmunization with CII in CFA revealed a normal splenic
architecture of the white and red pulp in both IFN-R KO and
wild-type mice. However, in IFN-R KO mice the red pulp was
extensively expanded due to extramedullary hemopoiesis with increased
numbers of megakaryocytes and erythropoietic and myelopoietic cells
(Fig. 4, A–D). Cytospin
preparations (Fig. 4, E and F and Table II) showed the occurrence in IFN-R KO
mice of much larger proportions of immature elements of the
monocyte-macrophage and granulocytic lineages than in wild-type mice.
The net numbers of T, NK, and NKT cells, as determined by FACScan, were
not different from those in similarly immunized wild-type mice. As a
contrast, Mac-1+ splenocytes expanded in a
significantly different way in IFN-R KO than in wild-type mice (Fig. 5A). In wild-type mice, the
total number of Mac-1+ cells increased for only 5
days, subsequently decreased and increased again from day 35 to reach a
maximum level of 12.8 x 106 on day 41. In
IFN-R KO mice, the increase in the number of
Mac-1+ spleen cells was significantly more
pronounced as it reached a maximum of 36.4 x
106 cells at an earlier time point (day 21). At
that point, the Mac-1+ cell population
represented 47.7 ± 4.0% of total splenocytes, against 10.2
± 2.3% in wild-type mice (n = 6). Interestingly, in
both strains of mice, the maximal increase in the
Mac-1+ cell population coincided with disease
onset as noted in previous experiments (Fig. 5A and Table I). The earlier and more pronounced increase in the splenic
Mac-1+ cell population in IFN-R KO mice thus
correlates with accelerated and increased disease incidence in
these mice.
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R KO and wild-type mice, respectively). Their number then
reverted to basal level and never was significantly different between
the mutant and wild-type mice (data not shown).
An increased Mac-1+ cell population was also seen
in wild-type mice immunized with CII in CFA that had been treated on
days 0, 7, and 14 with anti-IFN- Abs (Fig. 5B). As we
reported earlier (19), such treatment causes an
accelerated onset and increased disease incidence similar to that
observed in the mutant mice.
Treatment with anti-IL-12 or anti-IL-6 Ab inhibits both Mac-1+ splenocyte expansion and development of CIA
Increased numbers of immature myeloid cells (Table II and Fig. 4)
and enhanced expansion of the Mac-1+ cell
population following CFA-assisted immunization of IFN-R KO mice
(Fig. 5) might be due to the production of hemopoietic cytokines. Among
the possible candidates, we primarily focussed on IL-12. Our reason for
this choice was that the stimulatory effects of IL-12 on hemopoiesis
are counteracted by IL-12-induced IFN- (23) in much the
same way as IL-12 and IFN- affect the pathogenesis of CIA in
opposite directions (24). We also considered IL-6 as a
possible candidate because we found circulating levels of IL-6 to be
enhanced in IFN-R KO mice suffering from CIA (data not shown).
Hence, we studied the effect of treatment with anti-IL-12 or
anti-IL-6 Abs on both the expansion of Mac-1+
cells and on the development of arthritis. In each of two parallel
experiments, three groups of IFN-R KO mice were immunized with CII
in CFA (day 0) and were given i.p. Ab injections on days 0, 7, and 14
(anti-IL-12 or irrelevant IgG) or every other day from day 0 till
day 26 (anti-IL-6). Groups of naive IFN-R KO mice were included.
In the first experiment, two mice of each group were sacrificed on days
17, 22, and 36 to analyze the splenocyte population. In the second
experiment, mice were evaluated for signs of arthritis. Fig. 6A shows that anti-IL-12
Ab blocked and anti-IL-6 inhibited expansion of the
Mac-1+ population (day 22). Fig. 6B
shows that, in parallel with the effect on the
Mac-1+ population, treatments with the Abs
blocked (anti-IL-12) or inhibited (anti-IL-6) development of
the disease.
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R defect coordinately affects the Mac-1+
cell population and DTH reactivity against CII
To substantiate and mechanistically clarify the connection between
the Mac-1+ cell population and arthritis
development, we compared DTH development to CII in IFN-R KO mice
with that in wild-type mice, both after immunization with CII in
IFA or CFA. DTH reactivity was tested by the footpad swelling test on
day 21, i.e., at the time of maximal expansion of the
Mac-1+ cell population in the IFN-R KO mice.
Fig. 7 shows that, at this early time
point in pathogenesis, DTH development was restricted to IFN-R KO
mice that had received the CFA-assisted immunization schedule. This
observation strengthens the concept of a relation between expansion of
the Mac-1+ population, development of DTH for
CII, and joint involvement.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). However, both clinically and
histologically the arthritic lesions were similar whether CFA or IFA
was used. Here, as in a previous study, we have analyzed the role of
IFN- in CIA by comparing the disease course in IFN-R KO mice with
that in wild-type counterparts. We confirmed that ablation of
endogenous IFN- aggravates CIA induced by immunization of the mice
with CII in CFA. Significantly, however, we found that the opposite is
true in CIA induced by an IFA-assisted immunization schedule. Under
these conditions IFN-R ablation inhibited disease progression, thus
revealing a disease-promoting role of IFN-. Apparently, under the
influence of mycobacteria from the CFA, a pathogenic pathway becomes
dominant, which is counteracted by endogenous IFN-, resulting in the
disease-mitigating effect of IFN-.
Inhibition of CIA by ablation of IFN- in mice immunized without
assistance of mycobacteria was associated with suppression of both
anti-CII Ab and DTH reactivity at the time of arthritis
development. Therefore, in principle the disease-promoting component of
IFN-’s action may be mediated by enhancement of both cellular and
humoral immunity effector mechanisms. However, in our previous study
using the CFA-assisted schedule (19), we reported that Ab
formation was suppressed in IFN-R KO mice, despite their increased
disease scores. As shown here, DTH-responsiveness following
CFA-assisted immunization was augmented by the IFN-R KO mutation, an
effect that correlated well with enhanced arthritis development in
these mice. When IFA was used to induce CIA, DTH was significantly
lower in the mutant than in the wild-type mice. Therefore, it would
appear that augmented disease activity in the IFN-R-deficient mice
is mainly due to an increased effector function of cellular rather than
humoral immunity.
Our current study establishes the cellular effector function in CIA
that is inhibited by IFN-. Enhanced CIA in IFN-R KO mice
immunized by the CFA-assisted schedule was associated with pronounced
splenomegaly, extramedullary hemopoiesis, and overexpansion of a
Mac-1+ cell population. In these
IFN-R-deficient mice, the splenic Mac-1+ cell
population not only reached a higher maximal level than in wild-type
mice, but also did so at an earlier time point, which coincided with
the time of disease onset. In wild-type mice, the expansion of the
Mac-1+ cell population was less pronounced and
also followed a biphasic time course. Remarkably, the second peak
coincided with the later time of disease onset in these mice.
A key role for hemopoiesis in the pathogenesis of CIA is evidenced by
the observation that GM-CSF-deficient mice are protected against the
disease (26). Also, inhibitory effects of IFN- on
hemopoiesis have been documented in several systems. The stimulatory
effect of IL-12 on hemopoiesis was reported to be counteracted by
IFN- (23), and production of CSF-1 by monocytes was
found to be inhibited by IFN- (27). Of special
relevance is a study demonstrating a dramatic effect of the IFN-
knockout mutation on hemopoietic remodeling during infection with
Mycobacterium tuberculosis bacillus Calmette-Guérin.
The normal splenic architecture of the IFN- knockout mice was found
to be effaced by expanding myeloid cells (28). Our
observations indicate that a similar process takes place in IFN-R KO
mice that receive immunization schedules employing killed mycobacteria
as a component of CFA. Macrophages of IFN--deficient mice may
perform poorly in destroying the mycobacterial cell bodies. This may
result in long-term persistence of these bodies in the lymphoid system
and hence also in persistent stimulation of hemopoietic cytokines. Two
candidate hemopoietic cytokines, IL-6 and IL-12, were considered in
particular. We found that treatment of the mice with neutralizing
anti-IL-6 or anti-IL-12 Abs inhibited both the expansion of the
Mac-1+ cell population and the development of
arthritis. In accordance with our results, IL-6-deficient mice have
been reported to be resistant against CIA (29). IL-6 is
known to synergize with IL-3 (multi-CSF) in supporting the formation of
multilineage blast cell colonies in murine spleen cell cultures and
also to stimulate differentiation of myeloid cell lines into
macrophage- and granulocyte-like cells (for review see Ref.
30). Hence requirement of IL-6 for CIA development may be
due to its hemopoietic potential. IL-12 is another cytokine known to
affect hemopoiesis. Specifically, in IFN-R KO mice IL-12
administration caused rather increased bone marrow hemopoiesis and
strong extramedullary hemopoiesis in the spleen (23). Our
observation that anti-IL-12 Ab blocks expansion of the
Mac-1+ splenic population in IFN-R KO mice is
in line with these observations. These results, obtained with
anti-IL-6 and anti-IL-12 Ab treatments, are in accordance with
the concept that endogenous IL-6 and IL-12 are instrumental in bringing
about expansion of the Mac-1+ population, which,
in turn, contributes to the development of arthritis lesions.
Why are Mac-1+ cells so important in the
pathogenesis of CIA? Cells of the monocyte/macrophage lineage are known
to play an important role as producers of various cytokines and other
inflammatory mediators. It seems possible that the increased
availability of these cells specifically in IFN-R KO mice with CIA
induced by CFA-assisted immunization leads to a higher level of
infiltration of these cells into joints and thereby to a higher level
of inflammatory and destructive changes. The crucial role of
macrophages in destructive autoimmune diseases and the ability of
IFN- to keep this cell population in check received support from
other studies. Thus, administration of anti-Mac-1 mAb reduced the
severity of CIA in DBA/1 mice and prevented its adoptive transfer to
SCID mice (31). Furthermore, mice deficient in both B and
T cell functions, in spite of their inability to mount Ag-specific
immune responses, were found to be sensitive to induction of CIA,
leading the authors to the conclusion that lymphocyte-independent
pathogenic mechanisms operate in this model (32). Also, in
bacterial cell wall-induced arthritis in rats, exogenous IFN-
was reported to exert a protective effect, which correlated with an
inhibitory effect of IFN- on chemotactic responsiveness of
mononuclear phagocytes, apparently resulting from reduced expression of
C5a receptors accompanying enhanced expression of Ia Ag
(33). Finally, in experimental allergic encephalomyelitis,
a model in which CFA is used for induction and in which endogenous
IFN- protects against disease, the Mac-1+ cell
population was shown to play an essential pathogenic role (34, 35).
Collectively, our data indicate that 1) the role of the mycobacterial
component of CFA consists in stimulating myelopoiesis, potentially
leading to expansion of Mac-1+ cells that act as
DTH and arthritogenic effectors, and 2) that endogenous IFN-
normally restrains the expansion of this population. When mycobacteria
are omitted from the induction schedule, the stimulus for myelopoiesis
is significantly reduced. Therefore, one might speculate that, under
these circumstances, the inhibitory effect of IFN- on the number of
Mac-1+ cells may be of less consequence for
disease activity, while its activating effect on the aggressiveness of
these cells and on Ag-specific reactivity of T and B cells becomes the
dominant pathogenic factor. However, further investigation is needed to
obtain evidence for these views.
Our results have important implications for the clinical relevance of
the CIA model. The classical CIA model, which relies on CFA-assisted
immunization, is likely to represent clinical forms of arthritis
involving bacterial infections or other exogenous factors that
stimulate myelopoiesis. CIA induced without the help of mycobacteria
may be representative of clinical situations in which the myeloid
effector population plays a less prominent role. In rheumatoid
arthritis, the role of myeloid effector cells may vary from one time to
another or from one patient to another. This may explain why in
clinical trials systemic treatment with IFN- has yielded variable
results ranging from no effect to transient improvement (36, 37)
Our data also provide a possible explanation for protective effects of
IFN- in experimental autoimmune diseases other than CIA. Examples
are experimental autoimmune encephalomyelitis (10, 11, 12, 13, 14, 15, 16) and
experimental autoimmune uveitis (17, 18). So far, a
satisfactory explanation for protection by IFN- in these models has
not been offered. However, these models likewise rely on the use of CFA
for immunization, and ablation of IFN- has been shown to be
associated with increased influxes of monocytes/macrophages and
neutrophils in the lesions (14, 15, 18). Therefore, it
seems likely that the protective effect of endogenous IFN- in these
models, like in CIA, resides in its inhibitory effect on myelopoiesis
induced by the mycobacterial component of CFA. Finally, by revealing a
central role for myeloid cells in the pathogenesis of CIA, our results
identify myelopoiesis as a potential target for novel antirheumatic
drugs that has so far not been sufficiently explored.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Patrick Matthys, Rega Institute, University of Leuven, Laboratory of Immunobiology, Minderbroedersstraat 10, B-3000 Leuven, Belgium. E-mail address:
3 Abbreviations used in this paper: CIA, collagen-induced arthritis; CII, collagen type II; DTH, delayed-type hypersensitivity; IFN-R KO, IFN-R knockout.
Received for publication May 20, 1999. Accepted for publication July 7, 1999.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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