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The pathogenesis of group a streptococcal cell wall-induced polyarthritis in the rat comparative studies in arthritis resistant and susceptible inbred rat strains.

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1442
THE PATHOGENESIS O F GROUP A
STREPTOCOCCAL CELL WALL-INDUCED
POLYARTHRITIS IN THE RAT
Comparative Studies in Arthritis Resistant and Susceptible
Inbred Rat Strains
RONALD L. WILDER, JANICE B. ALLEN, LARRY M. WAHL.
GARY B. CALANDRA, and SHARON M. WAHL
This report addresses the host mechanisms which
regulate susceptibility and resistance to group A streptococcal cell wall-induced polyarthritis in the rat. We
have compared cell wall tissue distribution and persistence following a single intraperitoneal injection of an
aqueous suspension of the cell walls into arthritis susceptible L E W B and resistant F344/N female rats. Selected
aspects of the induced inflammatory reaction were also
compared. Our results suggest that development of
chronic erosive polyarthritis in this model is dependent,
in variable degrees, upon 1) deposition and persistence
of the cell walls in synovial tissues, and 2) a relative
inability of arthritis susceptible rats to neutralize the
proinflammatory properties of the cell walls.
Bacterial cell wall peptidoglycans possess many
proinflammatory properties relevant to the rheumatic
diseases. Cell walls are toxic in that they are capable
Presented in part at the VIII Pan-American Congress of
Rheumatology, Washington, DC. June 1982.
From the Arthritis and Rheumatism Branch, National Institute of Arthritis, Diabetes, Digestive and Kidney Disease and the
Laboratory of Microbiology and Immunology, National Institute of
Dental Research, National Institutes of Health, Bethesda, Maryland.
Ronald L . Wilder, MD, PhD: Senior Investigator, Arthritis
and Rheumatism Branch, NIADDK; Janice B. Allen, BS: Chemist,
Arthritis and Rheumatism Branch, NIADDK; Larry M. Wahl, PhD:
Research Biologist, Laboratory of Microbiology and Immunology,
National Institute of Dental Research; Gary B. Calandra, MD, PhD:
Medical Director, Merck Sharp Dohme Laboratories; Sharon M.
Wahl, PhD: Chief, Cellular Immunology Section, National Institute
of Dental Research.
Address reprint requests to Ronald L. Wilder, MD. PhD,
Arthritis and Rheumatism Branch, National Institute of Arthritis,
Diabetes, Digestive and Kidney Disease, National Institutes of
Health, Building 10, Room 9N228, Bethesda, MD 20205.
Submitted for publication March 7, 1983: accepted in
revised form June 23, 1983.
Arthritis and Rheumatism, Vol. 26, No. 12 (December 1983)
of stimulating or modulating immune or inflammatory
mechanisms by either direct or indirect means. For
example, they may initiate acute inflammation by
direct activation of the alternative pathway of the
complement system (1). They may also directly activate macrophages (immunoadjuvant activity) and indirectly modulate the activity of T and B lymphocytes,
fibroblasts, and other cells involved in acute and
chronic inflammation (2-7). Moreover, these polyclonal activators may also preferentially induce autoantibodies such as rheumatoid factor (8-9).
The in vivo biologic effects of a single administration of an aqueous suspension of purified bacterial
cell wall peptidoglycan, as well as that of the synthetic
peptidoglycan fragment, muramyl dipeptide, are transient (9-10). The transient biologic effects apparently
reflect rapid in vivo degradation and elimination of the
peptidoglycan (1 1). However, intact bacterial cell
walls consist of peptidoglycan linked to a complex
polysaccharide heteropolymer. In selected bacteria,
such as group A streptococci and other bacteria, the
polysaccharide heteropolymer appears to protect the
peptidoglycan from in vivo degradation and to enhance its biologic effects (12-14).
Relevant to this is the demonstration that a
single intraperitoneal injection of an aqueous suspension of peptidoglycan-polysaccharide fragments from
the cell walls of group A streptococci into experimental animals, particularly the rat, may induce chronic
polyarthritis which clinically, histologically, and radiologically resembles rheumatoid arthritis (14-17). The
arthritis is symmetric, involves peripheral and not
axial joints, and is characterized by extensive synovial
proliferation along with marginal joint erosions. In
contrast, a single intraperitoneal injection of an aque-
STREPTOCOCCAL CELL WALL-INDUCED POLYARTHRITIS
o u s suspension of purified peptidoglycan does not
induce arthritis (18).
The role of host factors in the pathogenesis of
this experimental model remains incompletely defined. Previously, we have shown that arthritis susceptibility in rats is strongly dependent upon strain and
hormonally mediated sex linked effects (16,17-19). For
example, LEW/N female inbred rats develop severe
polyarthritis with 100% incidence, whereas L E W/ N
males and F344/N males and females are relatively
resistant.
W e have hypothesized that the differences in
arthritis susceptibility reflect one or more of the following factors: 1) differences in the rate of degradation
or detoxification of the cell walls, 2) differences in the
amount of the cell wall fragments which disseminate
and deposit in various tissues, o r 3) differences in the
type, intensity, or duration of the inflammatory reaction induced by the cell wall fragments. In the present
study, we have addressed these parameters in an effort
t o further define general mechanisms which regulate
arthritis susceptibility and resistance in this model. We
have demonstrated group A streptococcal cell wall
antigens in the inflamed synovial tissues from L E W/ N
females but not in the normal appearing synovial
tissues from F344/N females. In both strains, however, cell wall antigens were demonstrated in other
tissues, particularly liver and spleen, for prolonged
periods following injection. W e also observed that
acute inflammatory lesions were induced at the sites of
cell wall deposition in both rat strains, but LEW/N
females developed more prominent chronic inflammatory lesions despite the presence of similar amounts of
cell wall antigen in both tissues.
MATERIALS AND METHODS
Animals. Specific pathogen-free inbred LEW/N and
F344/N female rats were obtained from the Small Animal
Section, National Institutes of Health. The animals weighed
approximately 100 gm at the initiation of each experiment.
Induction of arthritis. The preparation of peptidoglycan-polysaccharide fragments from group A streptococcal
cell walls and the induction and scoring of the chronic
polyarthritis were done as previously described in detail
(16). In brief, a sterile aqueous suspension, phosphate buffered saline (PBS), pH 7.4, of sonicated streptococcal cell
walls was injected intraperitoneally into both arthritis susceptible LEW/N females and relatively arthritis resistant
F344/N females at a dose equivalent to 20 pg of cell wall
rhamnose per gm body weight. Control animals were injected with an equal volume of PBS.
Although doses of cell wall fragments as much as 10fold smaller may induce chronic arthritis in LEW/N females,
1443
a latent period of 3 months or more may occur before
arthritis is apparent. In addition, the severity of arthritis is
more variable at lower doses. A dose of 20-60 pg of cell wall
rhamnoselgram body weight reproducibly induces severe
chronic arthritis after intraperitoneal injection within 2-6
weeks in young LEW/N females (4-9 weeks old). This dose
rarely induces chronic arthritis in F344/N inbred rats. Intravenous administration of sonicated cell wall fragments appears to require substantially less cell wall material, but we
have not systematically studied this route of administration
(Wilder et al: unpublished observations).
Histologic evaluation. Tissue specimens were fixed in
10% buffered formalin, decalcified in formic acid-sodium
citrate, if necessary, and stained with hematoxylin and eosin
(16).
Immunofluorescence. Fluorescein conjugated rabbit
globulin specific for group A streptococcal cell walls was
obtained from Difco, Detroit, MI. It had been cross-absorbed by the supplier to remove cross-reactivity to those
antigens which are shared between streptococcal groups A.
C, and G. Fluorescein conjugated rabbit globulin (Difco) was
used as a control. Both preparations were absorbed on rat
liver powder (Sigma, St. Louis. MO) before use. The 2
reagents were used at t h e same protein concentration (0.1
pg/ml) and had similar fluorescein-to-protein molar ratios
(-3). Immunofluorescence microscopic studies were performed on tissue smears by modification of the technique
described by Cromartie et al (14). Briefly. smears from
various tissues from streptococcal cell wall-injected or saline-injected control animals were air dried. fixed in methanol for 1 minute, stained in a humid chamber for 30 minutes,
and washed twice with PBS. Controls included smears from
saline-injected animals stained as above and smears stained
with fluorescein conjugated nonspecific rabbit immunoglobulin. Positive preparations appeared as cytoplasmic granular
apple green fluorescent particles of variable intensity. A11
controls were negative for particles with this appearance.
Specificity was also verified by absorption studies on group
A streptococcal walls. This absorption step completely
eliminated positive staining.
Preparation and characterization of mononuclear
cells. Single cell suspensions of spleen from animals injected
with streptococcal cell wall and saline were prepared under
aseptic conditions by mincing, followed by lysis of red blood
cells with 0.14M ammonium chloride and filtration through
sterile gauze. The cells were washed thoroughly and suspended in an appropriate medium (see below) for further
characterization. Viability was always greater than 95% as
judged by trypan blue exclusion.
The cells were counted and sized on a ZBI Coulter
counter integrated with a Channelyzer (Coulter Electronics,
Inc., Hialeah, FL). Macrophages were enumerated by latex
ingestion or nonspecific esterase staining (20).
Prostaglandin E2 production. Spontaneous in vitro
production of prostaglandin Ez (PGE2) by splenic mononuclear cells was determined as follows. Single cell suspensions of spleen (5 x lo6) were cultured at 37°C in 1 ml of
serum-free Dulbecco-Vogt’s medium (DMEM) (HEM Research, Rockville, MD) containing 100 unitdm1 penicillin,
100 pdml streptomycin, and 2 mM glutamine (DMEM) in 24well flat bottom plates (Costar, Cambridge, MA). Culture
1444
WILDER ET AL
4 DAYS
6 WEEKS
15 WEEKS
Figure 1. Demonstration of group A streptococcal cell wall antigens in the spleens of LEW/N female rats. The top row of photomicrographs
shows specific cytoplasmic immunofluorescent staining of spleen cell smears obtained 4 days, 6 weeks, and 15 weeks after cell wall injection
(original magnification X 850). The lower row shows corresponding darkfield views.
supernatants were collected after 24 hours. The amount of
PGEz produced was determined by radioimrnunoassay as
previously described (21). Results are given as the mean
PGEz productionkulture in 24 hours.
Monocyte chemotactic factor production. Spontaneous in vitro production of rnonocyte chemotactic factor (22)
was determined in the following manner. Single cell suspensions of spleen (2 x lo6) were cultured at 37°C in I ml of
serum-free DMEM in 24-well flat bottom culture plates.
Culture supernatants were collected after 48 hours. The
amount of monocyte chemotactic factor was quantitated, as
previously described, using glycogen elicited normal rat
peritoneal macrophages in 48-well microchemotaxis chamber$ (23). Endotoxin activated rat serum and culture media
alone served as positive and negative controls. The chambers were incubated for 2 hours at 37"C, and the filters were
then removed from the chambers, fixed, and stained. The
number of macrophages which migrated through the filters
was quantitated with an image analyzer integrated with a
Hewlett Packard 9815AIS Calculator (Optomax, Hollis,
NH). The data represent the mean number of macrophages
which migrated through the filter in triplicate assays.
Fibroblast activating factor production. Spontaneous
in vitro production of fibroblast activating factor by splenic
mononuclear cells was determined as previously described
(5). Briefly, spleen cells (2 x lo6) were cultured for 48 hours
at 37°C in 1 ml of serum-free DMEM in a 24-well flat bottom
plate. Culture supernatants were harvested, diluted 1:4with
media, and added to 5 x lo4 guinea pig fibroblasts for 48
hours at 37°C. The cultures were pulsed 4 hours before
harvest with 1 pCi/ml tritiated thymidine (TDR3H)
(Schwarz-Mann, Orangeburg, NY), specific activity 6 Ci/mmole. The data represent the mean counts per minute of
tritiated thymidine incorporated in triplicate assays.
The ability of mononuclear cell supernatants to induce fibroblast proliferation is not species specific (24,25).
Because of the availability and facility of obtaining guinea
pig dermal fibroblast populations, these cells were primarily
used as target cells for supernatant activity. Rat fibroblasts,
however, yield equivalent results.
Lymphocyte proliferation. The proliferative capability of single cell suspensions of spleen was studied by
culturing cells ( 106/ml)in DMEM containing 5% heat-inactivated normal rat serum and 5 x lO-'M 2-mercaptoethanol in
96-well flat bottom culture plates (Costar). Culture stimuli
were phytohemagglutinin (Burroughs Wellcome, Greenville,
NC) and concanavalin A (Calbiochem-Behring Corporation,
San Diego, CA). All stimuli were tested at a previously
determined optimal, a lower, and a higher dose. Cultures
were incubated for 3 days. Following a 4-hour pulse with 0.5
pCi/ml of TDR3H, the cells were harvested and the incorporation of thymidine was determined in a liquid scintillation
counter. The results are presented as the mean of TDR'H
incorporated in triplicate assays.
RESULTS
Tissue distribution and persistence of group A
streptococcal cell wall antigens. Previously published
comparative clinical and histopathologic analysis of
arthritis susceptible LEW/N and relatively resistant
F344/N female rats after intraperitoneal injection of
group A streptococcal cell walls demonstrated differences in the distribution of induced tissue abnormalities (16). To better define the mechanisms underlying
STREPTOCOCCAL CELL WALL-INDUCED POLYARTHRITIS
4 DAYS
6 WEEK5
1445
15 WEEKS
Figure 2. Demonstration of group A streptococcal cell wall antigens in the spleens of F3441N female rats. The top row of photomicrographs
shows specific cytoplasmic immunofluorescent staining of spleen cell smears obtained 4 days, 6 weeks, and 15 weeks after cell wall injection
(original magnification x 850). The lower row shows corresponding darkfield views.
these differences, the tissue persistence and distribution of the cell wall antigens after intraperitoneal
injection were compared by immunofluorescence
techniques.
The results of this analysis are presented in
Figures 1-3. As shown in Figures I and 2, cell wall
antigens were detected in the spleens of both rat
strains as early as 4 days or as late as 15 weeks or
longer after cell wall injection. These findings indicated that the relatively arthritis resistant F344/N females
did not appear to eliminate the cell wall antigens from
sites of tissue deposition more rapidly than arthritis
susceptible LEW/N females.
The distribution of cell wall antigens to synovial
tissues, however, did appear to differ significantly. As
demonstrated by the example in Figure 3, cell wall
antigens were consistently detected by immunofluorescence techniques in the cytoplasm of cells derived
from diseased synovial tissues from LEW/N females
with polyarthritis examined 4 days, 6 weeks, and 15 or
more weeks following cell wall injection. Cell wall
antigens, in general, were not detected in cells from
synovial tissues from arthritis resistant F344/N females. However, occasional animals (-5%) developed
mild acute arthritis which resolved within 7 days. We
occasionally detected equivocal evidence of cell wall
antigens in cells derived from these acutely inflamed
synovial tissues 4 days after injection, but generally
not at later times (data not shown).
The tissue distribution of cell wall antigens in
LEW/N females paralleled the sites of clinical and
histopathologic abnormalities cited above, suggesting
that, at least in LEW/N females, a cause and effect
relationship existed between cell wall deposition and
development of chronic inflammation at the site of
deposition. As summarized in Table I , the data from
F344/N females indicated additional complexity. Although these animals developed variable degrees of
enlargement and mononuclear cell hyperplasia in the
spleen in response to cell wall antigen deposition, the
liver generally appeared normal or, at most, exhibited
only minimal or low-grade evidence of chronic mononuclear cell infiltration, despite the demonstration of
cell wall antigens in both of these organs. Thus,
LEW/N females, in contrast to F344/N females, appeared more susceptible to the induction of systemic
chronic inflammation as indicated by the development
of more prominent and widely disseminated chronic
inflammatory lesions.
Host inflammatory reaction to cutaneous injection of group A streptococcal cell walls. The above data
indicated that acute and chronic mononuclear cell
inflammation developed at the sites of cell wall deposition, at least in LEW/N females. To confirm this point
1446
WILDER ET AL
6 WEEKS
4 DAYS
15 WEEKS
w
0
zw
0
2
c
0
3
U
Figure 3. Demonstration of group A streptococcal cell wall antigens in the synovial tissue smears from inflamed peripheral joints (ankle and
wrist) of LEWIN female rats. The top row of photomicrographs shows specific cytoplasmic immunofluorescent staining of cells obtained 4
days, 6 weeks, and 15 weeks after cell wall injection (original magnification X 850). The bottom row shows corresponding darkfield views.
cutaneous cell wall injection. As shown in Figure 4,
over a period of 24 hours both inbred rat strains
developed an acute inflammatory lesion at the site of
skin injection, which evolved to a predominantly
and to further exclude a generalized unresponsive
state to the cell walls as a mechanism for arthritis
resistance in F344/N females, we examined the local
response of F344/N and LEWlN females following
Table 1. Tissue distribution and persistence of peptidoglycan-polysaccharide fragments from group
A streptococcal cell walls parallel the distribution of chronic inflammatory lesions
Time after injection*
Rat straidtissue
4 days
6 weeks
15 weeks
F344lN
Spleen
++++
++++
+++
Liver
+++
t i
+
Focal accumulation of
mononuclear cells
Synoviumt
+/-
0
0
None/normal
LEWIN
Spleen
++++
++++
+++
Liver
++++
+++
++
++
+
+
Synoviumt
Chronic histopathologic findings
Mononuclear hyperplasia,
occasional focal necrosis
Mononuclear hyperplasia,
occasional focal necrosis
Diffuse, non-caseating
granulomas
Chronic synovitis with
mononuclear infiltration,
marginal bone erosion
* Qualitative estimate of relative quantities of antigenic cell wall material in tissue based upon
immunofluorescence studies with fluorescein conjugated rabbit anti-group A streptococcal cell wall.
t From joints of lower extremity.
STREPTOCOCCAL CELL WALL-INDUCED POLYARTHRITIS
1447
Figure 4. Cutaneous inflammatory responses of LEWM and F344/N females after injection of sonicated group A streptococcal cell walls (100
pg of cell wall rhamnose in 0.1 ml of phosphate buffered saline). A , C . and E show the response at 24 hours, 72 hours, and 2 weeks, respectively,
in LEW/N females. B, D, and F show the response at 24 hours, 72 hours, and 2 weeks, respectively. in F344/N females (original magnification
x 130).
mononuclear inflammatory infiltrate at 72 hours. Similar findings were noted after cell wall injection into the
footpads (data not shown). Of particular note, after 2
weeks the LEW/N females still exhibited significant
mononuclear cell infiltration at the site of injection.
but the F344/N demonstrated substantial resolution of
the lesion-only sparse residual mononuclear infiltration remained. In summary, the F344/N females responded acutely but appeared to resolve the lesions
more rapidly than the LEW/N females.
Inflammatory reaction to intraperitoneal injection of group A streptococcal cell walls. To obtain
additional evidence that the relative arthritis resistance observed in F344/N females was not trivially
explained by generalized unresponsiveness to streptococcal cell walls, we further characterized the inflammatory reaction at various times in the spleens of
injected LEW/N and F344/N females. As stated above
and previously published (16), the spleens from both
LEW/N and F344/N females displayed evidence of
WILDER ET AL
Table 2. Splenic mononuclear cells from group A streptococcal
cell wall (SCW) injected rats have an increased frequency of
macrophages relative to lymphocytes
Frequency of nonspecific esterase
positive cells andlor cells
ingesting latex beads (% of total
mononuclear cells)*
Source of mononuclear cells
F344lN spleen
Saline injected
SCW injected
LEW/N spleen
Saline injected
SCW injected
Table 4. Production of monocyte chemotactic factor by spleen cell
cultures of group A streptococcal cell wall (SCW) injected and
control rats
Macrophage chemotaxis (cellsloil
immersion field)*
~~
Mediator source
3 weeks after injection 6 weeks after injection
F344/N
Saline injected
SCW injected
LEWIN
Saline injected
SCW injected
Rat C5at
Media only
7.4 f 2
13.7 + 3
7.0 2 2
14.9 2 3
* Mean + standard deviation.
2 + 2
80 2 14
1 2 2
47 2 7
2 2 2
5 9 2 10
49 2 8
1 2 2
1 2
7 2
50 _t
1+
* Chemotaxis expressed as mean
t Endotoxin activated normal rat
clinical and histopathologic abnormalities including an
increase in weight and mononuclear cell number (1.5to 4-fold). The increase in mononuclear cell number
could be attributed to an increase in macrophages as
indicated by enumeration of cells which phagocytosed
latex particles or stained for nonspecific esterase, as
well as cell size profiling. The splenic mononuclear
cells from streptococcal cell wall injected animals
consisted of a higher frequency of macrophages relative to lymphocytes compared with saline injected
controls-about 7% in controls and about 14% in cell
wall injected animals, P < 0.01 (Table 2). The cytoplasm of the phagocytic cells from cell wall injected
animals exhibited extensive vacuolation compared
with saline injected controls (data not shown). These
abnormalities persisted for I5 weeks or longer. Reproducible differences between F344/N and LEW/N females by these criteria were not documented.
2 standard deviation.
serum, 1 :5 dilution.
To further characterize the splenic reaction, the
functional activity of mononuclear cells from both cell
wall and saline injected controls was assessed at
various times by several different assays (Tables 3-6).
Lymphocyte proliferation, as quantitated by tritiated
thymidine incorporation, in response to stimulation
with the mitogens concanavalin A and phytohemagglutinin from both cell wall and saline injected controls
was studied. A representative experiment is shown in
Table 3. Splenic mononuclear cells from both saline
injected LEW/N and F344/N female rats proliferated
well in reponse to these mitogenic stimuli. In contrast,
mononuclear cell proliferation from cell wall injected
animals was profoundly depressed at all mitogen doses
tested (P< 0.001). In parallel with our other findings,
the depressed splenic lymphocyte proliferation per-
Table 3. Proliferation to various stimuli of spleen cells from streptococcal cell wall (SCW) and saline
injected LEW/N and F344lN female rats
TDR3H incorporation,
cpmt
Stimulus'
None
Con A
PHA
0.1 pg/ml
0.5 &ml
1.0 pg/ml
5.0 pg/ml
10.0 pg/ml
0.1 pg/ml
0.5 pg/ml
1.0 &ml
5.0 pg/ml
10.0 @/ml
Saline injected
LE WIN
SCW injected
LEWIN
Saline injected
F344/N
SCW injected
F344lN
34
80
17,533
52,291
215,889
127,694
36
1.311
21,084
61,596
26, I60
97
69
1,070
468
3,634
1.172
44
480
2,343
130
70
170
365
31,106
49,745
130,204
118,810
523
6,254
38,995
95,227
41,001
86
I95
3,577
75 1
145
88
200
1,816
6,416
380
80
* Con A = concanavalin A; PHA = phytohemagglutinin.
t Mean incorporation of a representative study of spleen cells from individual rats. The variation in the
mean is within 2 10%. TDR'H
=
2
4
7
2
tritiated thymidine; cprn
=
counts per minute.
STREPTOCOCCAL CELL WALL-INDUCED POLYARTHRITIS
Table 5. Production of prostaglandin Ez by spleen cell cultures of
group A streptococcal cell wall (SCW) injected and control rats
PGE2 (nglculture) produced in 24 hours*
Mediator source
F344lN
Saline injected
SCW injected
LEW/N
Saline injected
SCW injected
* Mean
-t
3 weeks after injection 6 weeks after injection
886
4,142
2
?
54
303
1,081 2 104
1,415 2 86
1,679 2 774
6,094 f 1,107
705 2 21
1.822 ? 97
standard deviation.
sisted for 15 weeks or longer in both injected rat
strains. The key point of these findings was that both
rat strains developed evidence of a response, as reflected by an apparent functional abnormality, in comparison with saline injected controls.
In parallel with the lymphocyte proliferative
assays, the splenic production of several representative mononuclear cell derived inflammatory mediators was measured at several specific times (Tables
4-6). Table 4 shows a representative experiment
which demonstrates that splenic mononuclear cells
from cell wall injected animals from both rat strains
spontaneously produced greater amounts of monocyte
chemotactic factor in vitro than saline injected controls (P < 0.01). Table 5 shows a representative
experiment which demonstrates that these cells also
spontaneously produced greater amounts of prostaglandin E2 in vitro (P< 0.01). Similarly, Table 6 shows
that in vitro spontaneous production of fibroblast
activating factor was also increased after cell wall
injection, at least in F344/N females ( P < 0.01). The
production of these mediators was most easily demonstrated within the 6 weeks after injection. A consistent
finding, also evident in these data, was that within 6
weeks after injection, the arthritis resistant rat strain
generally produced greater amounts of these mediators. It was unclear whether these small differences in
the amount of mediator production between strains
had biologic significance, but of more importance,
these data provided additional confirmation that
F344/N females responded to group A streptococcal
cell walls. In fact, the data suggested, but did not
conclusively prove, that F344/N females responded
more vigorously than LEW/N females, at least as
measured by these assays.
DISCUSSION
These studies are part of our continuing effort
to understand the host factors that regulate suscepti-
1449
bility and resistance to group A streptococcal cell
wall-induced polyarthritis in the rat. Our strategy has
depended heavily on comparative analysis of highly
arthritis susceptible and relatively resistant inbred rat
strains, primarily the LEW/N and F344/N females,
respectively. In earlier studies, we demonstrated that
arthritis susceptibility was influenced by 2 or more
genetic loci, as well as by sex linked hormonal effects
(16,17-19). Consistent with these earlier conclusions,
the data presented here suggest that induction of
polyarthritis is dependent upon 2 major, possibly
interrelated defects: 1) dissemination and persistence
of the poorly degradable bacterial cell walls to synovia1 tissues of peripheral joints, and 2) induction of
chronic inflammation in these tissues as a consequence of a relative inability to neutralize the proinflammatory properties of the cell walls.
Following intraperitoneal administration, cell
wall antigens were readily demonstrated in phagocytic
cells in all sites of chronic inflammation examined in
LEW/N females, suggesting a causal relationship between tissue deposition of the cell walls and development of chronic inflammation at the site of deposition.
The findings in F344/N females further support these
conclusions but indicate that the regulation of susceptibility to the experimental polyarthritis is significantly
more complex. Inconsistent and equivocal evidence of
cell wall antigen deposition was observed in cells
derived from normal appearing synovium from these
animals. The failure to demonstrate cell wall antigens
in cells from these nondiseased tissues possibly reflects the absence of inflammatory cells, since cell
walls were only detected in diseased synovial tissues.
However, persistent cell wall antigens were clearly
demonstrated in the livers and spleens of the F344/N
females. Yet clinical and histopathologic evidence of
Table 6. Production of fibroblast activating factor by spleen cell
cultures of group A streptococcal cell wall (SCW) injected and
control rats
Fibroblast TDR3H
incorporation,
cpm*
Spleen cell supernatants
F344lN
Saline injected
SCW injected
LEWlN
Saline injected
SCW injected
Media only
* Mean 2 standard deviation. TDR’H
counts per minute.
5,711 2 358
12.602 f 1.004
7,085 -t 58
8,164 ? 812
3,556 -C 176
=
tritiated thymidine: cpm
=
1450
chronic inflammation in the liver consisted, at most, of
minimal mononuclear cell infiltration, in marked contrast to LEW/N females. The spleens from both strains
exhibited variable degrees of hypertrophy and mononuclear cell hyperplasia. These data, as stated above,
suggest that F344/N females, by an undefined mechanism, neutralize the proinflammatory properties of the
cell walls, despite their persistence in tissue.
Since it could also be proposed that F344/N
females are generally unresponsive to streptococcal
cell walls and consequently do not develop an inflammatory reaction, we examined several aspects of the
host response to the cell walls in both the LEW/N and
F344/N rats. Following cutaneous or footpad injection
of cell walls, both inbred rat strains developed acute
polymorphonuclear cell infiltration at the site within 24
hours, followed over the next 72 hours by mononuclear cell infiltration. These observations further demonstrate the proinflammatory potential of the cell walls
and exclude a complete unresponsive state in F344/N
females. Of particular note, after 2 weeks the lesions at
the site of injection were generally much less severe in
the F344/N. Although not all possible variables were
studied in this short term experiment, these findings
add additional support to the concept that F344/N
females efficiently neutralize the chronic inflammation-inducing properties of the cell walls.
Our studies also demonstrated that after intraperitoneal injection of cell walls both LEW/N and
F344/N females developed depressed splenic proliferative responses to mitogens concurrently with the production of several inflammatory mediators, including
prostaglandin EZ,fibroblast activating factor, and
monocyte chemotactic factor. Since the production of
these inflammatory mediators generally reflects cellular activation, these results further confirm that
F344/N females respond to streptococcal cell walls,
but their pathologic lesions are more limited. The
significance of the observation that the F344/N females consistently produced greater amounts of these
inflammatory mediators is unclear. The apparent T
cell mitogenic dysfunction is of additional interest. It is
general knowledge that this phenomenon is frequently
observed in chronic inflammatory states and is mediated, in part, by activated macrophages. Murine leprosy
with a heavy bacillary load is one example (26). Data
to be presented elsewhere have demonstrated that
activated macrophage dependent suppression also occurs in the streptococcal cell wall-induced arthritis
model and is not mediated by prostaglandins (Wahl S
et al: submitted for publication).
WILDER ET AL
In summary, our data support the view that
deposition of the poorly degradable peptidoglycan
containing cell walls, along with an inabiltty to neutralize their proinflammatory properties, leads to the
development of chronic inflammation at the site of
deposition. However, in view of the biphasic clinical
course of the experimental arthritis, it is conceivable
that the development of chronic disease reflects more
than a toxin-induced proinflammatory effect. Additional pathogenetic pathways, such as induction of an
autoimmune response or a hypersensitivity reaction,
could also play a role. These possibilities were not
addressed in the present studies. Comparative analysis
of these hypotheses in arthritis susceptible and resistant rat strains should further our understanding.
ACKNOWLEDGMENTS
We would like to thank Mr. Richard Whitt, Ms
Christine Winter, and Mr. William Helsel for their expert
technical assistance in several aspects of this work, and Ms
Susan Castillo for her assistance in preparing the manuscript.
REFERENCES
1. Greenblatt J, Boackle RJ, Schwab JH: Activation of the
alternate complement pathway by peptidoglycan from
streptococcal cell wall. Infect Immun 19:296-303, 1978
2. Takada H, Tsujimoto M, Kata K, Kotani S , Kusumoto
S, Inage M, Shiba T , Yano I, Kawata S , Yokogawa K:
Macrophage activation by bacterial cell walls and related synthetic compounds. Infect Immun 25:48-53, 1979
3. Nauciel C, Fleck J, Martin JP, Mock M, Nguyen-Hug
H: Adjuvant activity of bacterial peptidoglycans on the
production of delayed hypersensitivity and on antibody
response. Eur J Immunol 4:352-356, 1974
4. Wahl LM, Wahl SM, McCarthy JB: Adjuvant activation
of macrophage functions, Macrophage Regulation of
Immunity. Edited by ER Unanue, AS Rosenthal. New
York, Academic Press, 1980, pp 491-504
5 . Wahl SM, Wahl LM, McCarthy JB: Lymphocyte mediated activation of fibroblast proliferation and collagen
production. J Immunol 121:942-946, 1978
6. Saito-Tako T, Tanabe MJ, Mochizuki H, Matsumoto T,
Nakano M, Takada H, Tsujimoto M, Kotani S , Kusomoto S , Shiba T, Yokogawa K, Kawata S: Polyclonal B
cell activation by cell wall preparations of gram-positive
bacteria. Microbiol Immunol 24:209-218, 1980
7. Dziarski R: Polyclonal activation of immunoglobulin
secretion in B lymphocytes by staphylococcal peptidoglycan. J Immunol 125:2478-2483, 1980
8. Dziarski R: Preferential induction of autoantibody secre-
STREPTOCOCCAL CELL WALL-INDUCED POLYARTHRITIS
tion in polyclonal activation by peptidoglycan and lipopolysaccharide. I. In vitro studies. J Immunol 128:10181025, 1982
9. Dziarski R: Preferential induction of autoantibody secretion in polyclonal activation by peptidoglycan and lipopolysaccharide. 11. In vivo studies. J Immunol 128:10261030, 1982
10. Parant M, Parant F, Chedid L, Yapa A, Petit JF,
Lederer D: Fate of the synthetic immunoadjuvant, muramyl dipeptide (14-C-labelled) in the mouse. Int J Immunopharmacol 1:35-41, 1979
11. Schwab JH, Ohanian SH: Degradation of streptococcal
cell wall antigens in vivo. J Bacteriol94: 1346-1352, 1967
12. Gallis HA, Miller SE, Wheat RW: Degradation of 14-C
labelled streptococcal cell walls by egg white lysozyme
and lysosomal enzymes. Infect Immun 13: 1459-1466,
1966
13. Smialowicz RJ, Schwab JH: Processing of streptococcal
cell walls by rat macrophages and human monocytes in
vitro. Infect Immun 17591-598, 1977
14. Cromartie WJ, Craddock JG, Schwab JH, Anderle SK,
Yang CH: Arthritis in rats after systemic injection of
streptococcal cells or cell walls. J Exp Med 146:15851602, 1977
15. Clark RL, Cuttino JT, Anderle SK, Cromartie WJ,
Schwab JH: Radiologic analysis of arthritis in rats after
systemic injection of streptococcal cell walls. Arthritis
Rheum 22:25-35, 1979
16. Wilder RL, Calandra GB, Garvin AJ, Wright KD,
Hansen CT: Strain and sex variation in the susceptibility
to streptococcal cell wall-induced polyarthritis in the
rat. Arthritis Rheum 25: 1064-1072, 1982
17. Calandra GB, Wilder R, Garvin J , Hansen C, Wright K:
Genetic analysis of the development of group A streptococcal cell wall mediated arthritis in rats, Basic Concepts of Streptococci and Streptococcal Diseases. Edit-
18.
19.
20.
21.
22.
23.
24.
25.
26.
1451
ed by SE Holm, P Christensen. Surrey, England,
Reedbooks, Ltd., 1982, pp 313-314
Fox A, Brown RG, Anderle SK, Chetty C, Cromartie
WJ, Gooder H, Schwab JH: Arthropathic properties
related to the molecular weight of peptidoglycan-polysaccharide polymers of streptococcal cell wall. Infect
Immun 35: 1003-1010, 1982
Allen JB, Blatter D. Calandra GB, Wilder RL: Sex
hormonal effects on the severity of streptococcal cell
wall-induced polyarthritis in the rat. Arthritis Rheum
26:560-563, 1983
Yam LT, Li CY, Crosby WH: Cytochemical identification of monocytes and granulocytes. Am J Clin Pathol
55:283-290, 1971
Wahl LM: Production and quantitation of prostaglandins, Manual of Macrophage Methodology. Edited by
HB Herscowitz, HT Holden, JA Bellanti, A Ghaffar.
New York, Marcel Dekker, Inc., 1981, pp 423-429
Ward PA, Volkman A: The elaboration of leukotactic
mediators during the interaction between parental-type
lymphocytes and F, hybrid cells. J Immunol 115:13941399, 1975
Falk W, Goodwin RH Jr, Leonard LJ: A 48-well microchemotaxis assembly for rapid and accurate measurement of leukocyte migration. J Immunol Meth 33:239247, 1980
Wahl SM, Gately CL: Modulation of fibroblast growth
by a lymphokine of human T cell and continuous T cell
line origin. J Immunol 130:1226-1230, 1983
Wahl SM: Immunologically induced fibrosis, Progress in
Diseases of the Skin. Volume 2. Edited by R Fleischmajer. New York, Grune and Stratton (in press)
Yamaura N, Akiyama T, Nakano T: Mitogen induced
DNA synthesis in various mouse strains infected with a
large or small dose of murine leprosy bacilli. Microbiol
Immunol 25:245-255, 1981
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induced, group, streptococcus, strains, rat, pathogenesis, cells, susceptible, inbreds, wall, resistance, arthritis, polyarthritis, comparative, studies
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