INFECTION AND IMMUNITY, Apr. 1989, p. 1177-1185 Vol. 57, No. 40019-9567/89/041177-09$02.00/0Copyright © 1989, American Society for Microbiology

Protracted Anemia Associated with Chronic, Relapsing SystemicInflammation Induced by Arthropathic Peptidoglycan-Polysaccharide

Polymers in RatsR. BALFOUR SARTOR,l.2* SONIA K. ANDERLE,3 NADER RIFAI,4 DAVID A. T. GOO,4

WILLIAM J. CROMARTIE,3 AND JOHN H. SCHWAB2'3Department of Medicine,1 Department of Microbiology and Immunology,3 Department ofPathology,4 and Core Center

for Diarrheal Disease,2 University of North Carolina, Chapel Hill, North Carolina 27599-7080

Received 3 October 1988/Accepted 6 January 1989

Mild hypoproliferative anemia with abnormal iron metabolism frequently accompanies chronic inflamma-tion and infection in humans. To determine whether anemia is associated with chronic relapsing arthritisinduced by bacterial cell wall polymers, serial determinations of the hematocrit were measured in rats injectedintraperitoneally with sonicated peptidoglycan-polysaccharide fragments from group A streptococci. Acuteanemia peaked 5 to 10 days after injection, and chronic, spontaneously relapsing anemia persisted for 309 days.51Cr labeling demonstrated decreased erythrocyte survival, i.e., a half-life of 8.4 days in cell wall-injected ratsversus 11.8 days in controls. Erythrocytes were mildly microcytic, and leukocyte counts were elevated duringearly spontaneous reactivation of arthritis, 15 days after injection of peptidoglycan-polysaccharide. Bonemarrow myeloid/erythroid precursor ratios were elevated in arthritic rats (P < 0.0001). Purified peptidoglycanproduced an acute anemia lasting 10 days, while injection of group A streptococcal polysaccharide andmutanolysin-digested cell wall did not affect the hematocrit. The minimal effective dose of peptidoglycan-polysaccharide was 5 ,ug of rhamnose per g (body weight). Serum iron and transferrin levels were decreasedin cell wall-injected rats (P < 0.005) and were closely correlated with hematocrit values and joint inflammatoryscores. Stainable iron was increased in the liver, spleen, and mesenteric lymph nodes and unchanged in thebone marrow of cell wall-injected rats. Anemia accompanying chronic, relapsing systemic inflammationinduced by peptidoglycan-polysaccharide polymers appears to be an excellent animal model of the anemia ofchronic disease.

Mild nonprogressive anemia accompanies chronic inflam-mation of diverse etiologies, including chronic bacterial andfungal infections (42), rheumatoid arthritis (43), Crohn'sdisease (10), and widespread malignancies (13). The anemiaof chronic disease (inflammation) is characterized by mildlydecreased circulating erythrocyte (RBC) survival, lack of anappropriate bone marrow compensatory response, and dis-ordered iron kinetics (13, 24, 48). Typically, the serum iron,total iron-binding capacity (TIBC), and marrow sideroblastsare decreased in spite of normal-to-increased bone marrowand tissue iron stores. Recent studies have suggested thatmonokines secreted by activated macrophages may be re-sponsible for suppression of bone marrow erythroid precur-sors (33, 37). Anemia accompanies several experimentalinflammatory states associated with macrophage activation,including adjuvant arthritis (25), arthritis induced by viableBCG (26) and Mycoplasma arthritidis (11), and turpentineabscesses (32). These experimental models have been pro-posed as animal models of the anemia of chronic inflamma-tion, however, none of the models produce protracted ane-mia nor exactly reproduce the bone marrow suppression anddisordered iron metabolism found in the anemia of chronicdisease (48).

Bacterial cell wall polymers induce well-characterizedacute and chronic granulomatous inflammation in a varietyof organs after local and systemic injection (35, 41). Pepti-doglycan-polysaccharide (PG-APS) polymers derived from

* Corresponding author.

group A streptococci induce an acute and chronic systemicinflammatory response characterized by hepatic granulomas(20, 47) and spontaneously relapsing arthritis (8) that canpersist at least 1 year after a single intraperitoneal (i.p.)injection into susceptible rat strains. Macrophages are acti-vated to secrete increased amounts of interleukin-1 (IL-1) byboth in vitro and in vivo exposure to PG-APS (L. A. Bristoland J. H. Schwab, submitted for publication).

This study is designed to determine whether a single i.p.injection of PG-APS would produce protracted anemia andto determine whether this well-characterized and reproduc-ible chronic, systemic inflammatory response in geneticallysusceptible rats could serve as a reliable model of the anemiaof chronic disease. We demonstrate that PG-APS produces achronic, spontaneously relapsing anemia that persists atleast 10 months after a single i.p. injection and whose courseclosely parallels activity of peripheral arthritis. PG is themoiety of PG-APS responsible for the anemia and arthritis.PG-APS-induced anemia resembles human anemia ofchronic disease in the following respects: it is mild tomoderate, RBC survival is decreased, RBCs are slightlymicrocytic, serum iron and transferrin concentrations aredecreased, and reticuloendothelial iron stores are normal orincreased.(A portion of this work was presented as a poster at the

87th Annual Meeting of the American Society for Microbi-ology in Atlanta, Ga., 1 to 6 March 1987, and was publishedas an abstract [R. B. Sartor, S. K. Anderle, W. J. Cromar-tie, and J. H. Schwab, Abstr. Annu. Meet. Am. Soc. Micro-biol. 1987, B-146, p. 49].)

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1178 SARTOR ET AL.

MATERIALS AND METHODS

Animals. Female inbred Lewis rats were purchased fromCharles River Breeding Laboratories, Inc., Wilmington,Mass., and were housed 2 or 3 per cage with free access toPurina rat chow and water at all times. Rats weighed 140 to160 g at the time of injection.

Cell wall polymer preparation. PG-APS polymers wereisolated under aseptic conditions from the cell walls of strainD58 of group A streptococci (Streptococcus pyogenes) aspreviously described in detail (40). Briefly, streptococcigrown in Todd-Hewitt broth (BBL Microbiology Systems,Cockeysville, Md.) were disrupted in a Braun MSK cellhomogenizer (Bronwell Scientific, Inc., Rochester, N.Y.),and the cell wall fraction was separated by filtration andcentrifugation. The cell walls were extracted three times in2% sodium dodecyl sulfate (Sigma Chemical Co., St. Louis,Mo.) at 56°C and then washed extensively and dialyzed toremove the sodium dodecyl sulfate. Purified PG-APS poly-mers (400 mg) were suspended in 20 ml of sterile phosphate-buffered saline (PBS) (pH 7.2) and sonicated for 35 min usinga 0.5-in. (1.27-cm) probe and a setting of 8 (model 350;Branson Sonic Power Co., Danbury, Conn.). The suspen-sion was centrifuged at 10,000 x g for 30 min to remove largeparticles. PG-APS fragments prepared by this method havemolecular weights ranging from 5 x 106 to 5 x 108 (16).Sterility was confirmed by culturing 0.1 ml on sheep bloodagar. PG-APS concentrations for injections were calculatedon the basis of rhamnose values determined by the methodof Dische and Shettles (9). Immediately before injection,PG-APS was diluted to appropriate concentrations in PBS(pH 7.2) and sonicated for 3 min with a 9-kilocycle Sonicoscillator (Raytheon Co., Waltham, Mass.) to disperse ag-gregates.

Preparation of PG-APS fractions. Group-specific APS andPG were prepared by hot formamide treatment of cell walls.APS was purified from the supernatant by the method ofKrause (22), as modified by Chetty et al. (4), and contained47% rhamnose. PG was purified by multiple formamideextractions by the method of Krause and McCarty (23) andcontained 0.5% rhamnose.

Mutanolysin-digested PG-APS was prepared by a modifi-cation of the method of Chetty et al. (5). The M-1 fraction ofmutanolysin, an endo-N-acetylmuramidase derived fromStreptomyces globisporus, was obtained from Miles Labo-ratories, Inc., Elkhart, Ind. Samples (8 ml) of sonicatedPG-APS (27.4 mg of rhamnose) were diluted with 12 ml ofPBS containing 0.5 M sodium citrate and 0.05 mM MgCl2containing 3.29 mg of mutanolysin and then incubated at37°C with constant mixing for 11 h. The optical densitymeasured at 600 nm decreased from 0.492 to 0.135 afterincubation with mutanolysin. Mutanolysin-digested PG-APShas previously been shown to have a molecular weight of <5x 106 and to produce only transient edema without arthritisupon i.p. injection (5).

Labeling of erythrocytes with -5Cr. RBCs from Lewis ratswere labeled in vitro with 51Cr as previously described (34).51Cr-labeled RBCs were diluted to 1.07 x 10' cpm/ml in PBSand injected within 30 min.

Experimental design. Lewis rats were injected i.p. with 1ml of PG-APS or cell wall fraction diluted with PBS (pH 7.2)to the appropriate concentration (rhamnose concentrationscited in individual experiments). In each experiment, controlrats were injected i.p. with 1 ml of PBS. In the studymeasuring RBC survival, 0.5 ml of 51Cr-labeled RBCs (5.35x 105 cpm) was injected intravenously 30 min before i.p.

injection of PG-APS or PBS. Serial determinations of hema-tocrit (HCT), 51Cr counts per minute, or sedimentation rateswere obtained at intervals outlined in individual experimentsfrom blood derived from transection of the tip of the tail. Aminimal volume of blood was obtained to prevent irondeficiency. Bleeding was controlled by digital pressure onthe tail above the transection and by cauterization immedi-ately after blood collection. Blood was collected directly intotwo heparinized 75-mm-long microhematocrit capillarytubes (approximately 70 [LI of operational volume; FisherScientific, Pittsburgh, Pa.) for duplicate HCT and sedimen-tation rate determinations and into heparinized microcentri-fuge tubes (approximately 200-pL volume) for 51Cr or auto-mated cell counting. Serum for iron and transferrin levelswas obtained at necropsy by cardiac puncture. All injectionsand blood collections were done under ether inhalationanesthesia to minimize discomfort, and animals were killedby CO2 overdose.Measurement of laboratory and clinical parameters. HCT

values were determined in duplicate microhematocrit tubesimmediately after centrifugation at 12,600 x g for 3 min.Spontaneous sedimentation of RBCs was measured in mi-crohematocrit capillary tubes allowed to stand upright for 30min after collection at room temperature. The percentplasma relative to total volume was read with a microhema-tocrit capillary tube reader (Lancer, Brunswick Co., St.Louis, Mo.). The amount of 51Cr within 0.1 ml of heparin-ized blood was determined in a gamma counter (1197 series;Automatic Gamma Counting System; Searle Analytic, Inc.,Des Plains, Ill.). Counts per minute at each time point werecorrected for the spontaneous decay in 51Cr activity (phys-ical half-life, 27.7 days). An automated cell counter (150series; Baker Instruments Corp., Allentown, Pa.), pro-grammed for rat blood cells, was used for hemoglobin, RBCand leukocyte (WBC) counts, and mean corpuscular volumedeterminations. Transferrin was measured immunoturbidi-metrically (31) with the following modifications. Rat serawere diluted 1:25 with PBS. Rabbit anti-rat transferrinantiserum (Nordic Immunological Reagents, El Toro, Calif.)was diluted 1:50 with 40 g of polyethylene glycol per liter ofPBS. Within-run imprecision for the transferrin assay wasless than 5%. The iron concentration was determined byusing Beckman's ferrozine method (Beckman Instruments,Inc., Brea, Calif.) by the instructions of the manufacturer.The analytical imprecision for the iron assay was less than4%. The determinations of transferrin and iron were per-formed on the Cobas-BIO centrifugal analyzer (Roche Ana-lytical Instruments Inc., Nutley, N.J.). Iron stains wereperformed on femoral bone marrow touch preparations andFormalin-fixed liver and spleen sections by the method ofGomori (18). Bone marrow myeloid/erythroid precursorratios were performed on touch preparations of femoralmarrows, which were stained with Wright's-Giemsa re-agents. Bone marrow cellularity was determined in Forma-lin-fixed decalcified femurs, sternums, and ankle jointsstained with hematoxylin and eosin. Arthritis was measuredby using a clinical joint score of 0 to 4+ for each ankle(maximum score, 16) (8). We have previously demonstrateda close correlation of clinical joint scores with radiographic(6) and histologic (8) assessments of joint inflammation.

Statistical evaluation. Mean values of each experimentalparameter were calculated for each group of rats and com-pared by Student's unpaired t test. Experimental parameterswere correlated by linear regression analysis.

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ANEMIA INDUCED BY BACTERIAL CELL WALLS 1179

50

0tO 40+1C

30

0-

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200 oE 1I)I

40 80 120 160 200Days After Injection

.-I

7wcn

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FIG. 1. Acute and chronic anemia and arthritis induced by PG-APS polymers derived from group A streptococci. Sterile, sonicatedPG-APS (20 jig of rhamnose per g) or PBS was injected i.p. into 10 female Lewis rats per group. Serial determinations ofHCT were measuredfrom blood collected in duplicate microcapillary tubes after transection of the tail. Joint scores were measured clinically as specified in thetext. The joint score of PBS-injected rats was 0 (scores not shown). SEM, Standard error of the mean; SD, standard deviation.

RESULTS

Time course of anemia and arthritis. Serial HCTs weremeasured in female Lewis rats injected with PG-APS (20 ,ugof rhamnose per g) or PBS. PG-APS-injected rats developeda transient increase in HCT 17 h after injection, followed bya protracted anemia compared with the PBS-injected controlgroup (Fig. 1). The time course of anemia followed a typicalpattern in this and subsequent experiments. An acute drop inHCT occurred by 3 days after injection, with the lowestHCT values (36.2% in the PG-APS group versus 45.1% in thecontrol group [P < 0.0001]) recorded 10 days after injection.Over the next week, the HCT slowly increased but neverreached control values. Between 3 and 5 weeks after injec-tion, individual rats developed a spontaneous recurrence ofpersistent anemia. The peak of the chronic anemia at thisdose ofPG-APS occurred 49 days after injection (HCT valueof 36.4% ± 2.8% versus 46.5% ± 1.1% in the control group,[P < 0.0001]). HCT values in the PG-APS-injected rats weresignificantly lower than those of PBS-injected controls for309 days after injection. Arthritis, as measured by theclinical joint score, demonstrated a similar acute phase,which peaked 3 days after PG-APS injection, and a chronic,spontaneously relapsing phase beginning 4 weeks after in-jection (Fig. 1). Mean values obscure the occasional dra-matic change in the HCT and joint scores of individual ratsduring the spontaneous relapses and remissions of anemiaand arthritis. The sporadic nature of the recurrence ofanemia led to relatively high standard deviations of HCTvalues 3 to 4 weeks after injection (Fig. 1). All rats injectedwith PG-APS abruptly developed recurrent anemia (HCT ofs40% and a decrease in HCT by >3 percentage pointswithin 1 week); 57% (4 of 7) had a reactivation of arthritiswithin 1 week of the sudden drop in HCT. Recurrent anemiapreceded or accompanied spontaneous reactivation of arthri-tis, which in the Lewis rat persists indefinitely as an anky-losed joint with massive bony proliferation (20). Control ratsinjected with PBS developed neither anemia nor arthritis.RBC survival. Survival of syngeneic RBCs labeled in vitro

with 51Cr was compared in rats injected with PG-APS (20 ,ugof rhamnose per g [body weight]) or PBS. PG-APS-injectedrats had significantly lower counts per minute per 100 RI ofblood than the controls did, beginning 7 days after injection

(I)+1C 1000

E

D \ \~~~~~~~~~~*00

0

C)0.100,

10 20 30 40 50 60 70Days After Injection

FIG. 2. RBC survival following PG-APS or buffer injection.Shown are serial measurements of 51Cr activity in heparinized bloodfollowing intravenous injection of syngeneic RBCs (5.35 x i05 cpm)labeled in vitro 30 min before the i.p. injection of PG-APS (20 jig ofrhamnose per g) or PBS into groups of 10 female Lewis rats (thesame rats were used in Fig. 1). Results are corrected for thespontaneous decay of 51Cr activity. SEM, Standard error of themean.

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TABLE 1. Ability of PG-APS constituents to induce anemia

Material HCT" (%) (mean ± SEM) on day:injected 1 6 10 14 55

PG-APSb 45.6 ± 0.5* 40.5 ± 0.4** 41.6 ± 0.7* 42.3 ± 0.7* 44.5 ± 2.1PG-APS digestc 43.7 ± 0.4 43.0 ± 0.6 44.2 ± 0.6 43.2 ± 0.7 46.5 ± 0.3PGd 41.1 ± 0.6* 38.3 ± 0.9** 42.2 ± 0.8* 43.5 ± 0.8 46.9 ± 0.6APSe 44.2 ± 0.6 42.6 ± 0.4 45.1 ± 0.6 44.8 ± 1.2 46.8 ± 0.2PBSf 43.5 ± 0.4 44.0 ± 0.6 45.2 ± 0.7 44.7 ± 0.4 48.1 ± 0.2

a * for P < 0.01 compared with the PBS-injected group; **, P < 0.001 compared with the PBS-injected group.b PG-APS polymers from group A streptococci (30 ,ug of rhamnose; approximately 90 ,ug [dry weight] of PG-APS per g [body weight]) injected i.p. into six

female Lewis rats.c PG-APS polymers from group A streptococci (30 p.g of rhamnose per g [body weight]) predigested for 12 h with mutanolysin and then injected i.p. into six

rats.d Purified PG (45 p.g [dry weight]/g [body weight]) injected i.p. into six rats.e Formamide-extracted group A streptococcal APS (30 p.g of rhamnose per g [body weight]) injected i.p. into six rats.f PBS injected i.p. into six rats (buffer control).

and extending until 51Cr was no longer detectable (Fig. 2).The calculated half-life of blood cells, based on four half-lives, was 8.4 days in the PG-APS-injected rats comparedwith 11.8 days in controls. There was no evidence ofgastrointestinal blood loss by Hemoccult (Smith-Kline Diag-nostics, Sunnyvale, Calif.) testing of fecal samples and no

evidence of bleeding from the cauterized tails. These resultsdemonstrate a moderate decrease in RBC survival in PG-APS-treated rats which was not due to bleeding.

Active moiety of PG-APS. To determine which portion(s)of the PG-APS polymer was responsible for protractedanemia, we measured HCTs of rats injected with equivalenthigh doses of purified PG and APS as well as enzymaticallydegraded PG-APS (Table 1). As described above, rats in-jected with nondigested PG-APS polymers developed acuteand chronic anemia and arthritis after a transient increase inHCT 1 day after injection. Rats injected with PG hadarthritis and significantly decreased HCTs from 1 to 10 daysafter injection, with peak anemia at 6 days, but no evidenceof chronic anemia or arthritis. In contrast, rats injected withAPS or mutanolysin-digested PG-APS did not demonstratedecreased HCT values or arthritis at any time point but diddevelop transient peripheral edema 30 min after injection as

previously described (4, 5). PG-injected rats did not developedema, but four of six rats had clinically detectable diarrhea,and all six had conjunctivitis.Dose response. Rats were injected with PG-APS in doses

ranging from 0 to 40 p.g of rhamnose per g (body weight) todetermine the minimally effective dose that produced acuteand chronic, relapsing anemia (Table 2). Rats injected with

0.5 or 1 p.g of rhamnose per g (body weight) did not developclinically significant anemia at any time, although sporadictime points showed a small but statistically significant differ-ence in HCT compared with control values. PG-APS indoses between 10 and 40 p.g of rhamnose per g (body weight)induced acute anemia peaking at 4 to 7 days after injection ina dose-dependent fashion. Rats injected with 5 to 40 ,ug ofrhamnose developed chronic, spontaneously relapsing ane-mia with very interesting differences in the timing of therecurrences, depending on the dose injected (Table 3). Ratsreceiving 5 ,ug/g (body weight) i.p. had no acute change inHCT but developed chronic anemia 35 to 113 days afterinjection. Only one rat in this group had spontaneous reac-tivation of anemia. Rats receiving 10 to 20 I.g of rhamnoseper g (body weight) developed recurrent, chronic anemiawhich, in most animals, never returned to normal levels.These rats developed a recurrence of moderate (HCT of<40%) anemia 18 to 28 days after PG-APS injection; thesecond and third recurrences of anemia occurred 50 to 113days after injection. In contrast, HCTs of rats receiving thehighest dose of PG-APS (40 jig of rhamnose per g [bodyweight]) returned to completely normal levels by 10 daysafter injection and remained no different than control levelsuntil 70 days after injection (Tables 2 and 3). Eventually, fiveof nine rats injected with 40 jig of rhamnose PG-APS per g(body weight) developed moderate chronic anemia, but nonehad a second relapse during the 135-day observation period.

Iron and transferrin levels. To determine whether ironmetabolism was abnormal during chronic inflammation in-duced by PG-APS, serum iron and transferrin levels were

TABLE 2. Anemia with increasing doses of PG-APS polymers

PG-APS HCTb (%) (mean ± SEM) on day:

dose 7 14 22 50 113

0 44.3 ± 0.3 47.8 ± 0.2 46.0 ± 0.3 48.6 ± 0.3 47.3 ± 0.60.5 43.2 ± 0.4 46.5 ± 0.3* 46.0 ± 0.6 48.1 ± 0.4 47.3 ± 0.51 43.6 ± 0.5 46.8 ± 0.6 45.8 ± 0.3 47.5 ± 0.2* 46.4 ± 0.35 44.4 ± 0.3 46.8 ± 0.5 45.8 ± 0.3 43.9 + 1.9 42.1 ± 1.2*10 42.9 ± 0.4* 43.7 ± 0.8** 40.2 ± 1.9* 43.9 ± 1.9 42.9 ± 0.9*15 40.9 ± 0.8** 39.8 ± 1.3** 35.3 ± 1.6** 43.6 ± 1.2* 42.6 ± 0.5**20 41.9 ± 0.9* 42.2 ± 1.4* 39.0 ± 2.5 43.6 ± 1.2* 43.5 ± 1.1*40 39.6 ± 0.8** 46.0 + 0.3** 45.9 ± 0.4 48.7 ± 0.3 38.9 ± 1.5**

" PG-APS polymers from group A streptococci injected i.p. into six to nine female Lewis rats per group. Values are in micrograms of rhamnose per gram (bodyweight).

b *, P < 0.02 compared with rats given no PG-APS; **, P < 0.001 compared with rats given no PG-APS.

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ANEMIA INDUCED BY BACTERIAL CELL WALLS 1181

TABLE 3. Incidence and timing of severe anemia(HCT of <40%) with increasing doses of PG-APS

Chronic anemiaPG-APS Acute anemiadose' Recurrence 1 Recurrence 2

No.b Peak" No. Peak No. Peak

0 0 0 00.5 0 0 01.0 0 0 05 0 5/8 35-113 1/8 70

10 0 5/9 22-28 5/9 6315 4/7 7 7/7 22 4/7 63-7020 5/9 7-10 7/9 18-22 3/9 63-7040 5/7 4 5/9 70-113 0

a See footnote a of Table 2.b Number of rats with anemia/number of rats injected.' Peak occurrence of anemia. Values shown are days after i.p. injection of

PG-APS.

measured 36 days after i.p. injection of PG-APS (15 ,ug ofrhamnose per g [body weight]). PG-APS-injected rats hadsignificantly lower iron and transferrin levels than buffer-injected control rats (Table 4), despite one misinjected rat inthe PG-APS group which did not develop arthritis, anemia,or abnormal iron and transferrin levels.Bone marrows of rats injected with PG-APS 15 days prior

to sacrifice contained normal concentrations of intracellulariron by cytochemical staining. By 33 and 65 days afterinjection, iron staining within the bone marrow was dimin-ished compared with iron staining in controls but remaineddetectable. Iron-containing macrophages were markedly in-creased in livers of rats injected with PG-APS, with intenselystaining enlarged macrophages concentrated at the peripheryof hepatic granulomas (Fig. 3), portal tracts, and centralveins. Kupffer cells of PG-APS-injected rats frequentlycontained detectable iron and were randomly distributed. Incontrast, livers of control rats had rare Kupffer cells con-taining faintly staining iron, which were usually adjacent tocentral veins. Mesenteric lymph nodes and spleens of PG-APS-injected rats contained increased numbers of iron-containing macrophages, frequently present in aggregatesand giant cells.

Correlation of experimental parameters. PG-APS-injectedrats which developed anemia consistently displayed concur-rent arthritis, increased RBC sedimentation rates, hepaticgranulomas, and decreased serum iron and transferrin levels(Table 4). There was a striking temporal relationship be-

s W.f

'A I s; b ...\

FIG. 3. Iron-laden macrophages within a hepatic granuloma. Afemale Lewis rat was injected i.p. with PG-APS polymers (30 ,ug ofrhamnose per g) 65 days before necropsy. This small granuloma inthe hepatic parenchyma near a central vein contains macrophageswith densely staining iron by the method of Gomori (18). Magnifi-cation, x450).

tween arthritis and delayed onset or recurrent anemia (Fig.4) as well as between the acute phase of inflammation andanemia (Fig. 1). In pooled experiments, 35 of 39 (90%) ratswhich developed recurrent anemia had their first reactiva-tion of arthritis within 1 week of the abrupt drop in HCT. Atfour time points between 10 and 36 days after injection ofPG-APS or PBS, HCT and joint scores were very closelycorrelated, with r values ranging from -0.93 to -0.98 and P< 0.0001. Owing to variability in the timing of spontaneouslyreactivating inflammation within an experimental group,mean values did not adequately demonstrate the close cor-relation that existed between HCT values and joint scoresduring recurrence of arthritis and anemia in individual rats(Fig. 4). Lewis rats develop irreversible destructive arthritis

TABLE 4. Correlation of experimental parameters in cell wall-injected and control rats

Mean" + SEMMaterial injectedor correlation . Serum RBCcoefficient HCT (%) Serum Iron transferrin sedimentation Joint score

(jig/dI) ~~(mgldl)(%

Material injectedPG-APSb 38.8 ± 2.7* 149 ± 49* 30.4 ± 12.9* 3.8 ± 1.3** 6.4 ± 1.6*PBSC 47.7 ± 0.2 358 19 79.0 ± 1.5 0.2 ± 0.1 0 0

Correlation coefficientHCT 0.97 0.97 0.87 0.98Joint score 0.98 0.97 0.98 0.86Transferrin 0.97 0.95 0.68 0.98

a *, p < 0.005 compared with PBS-injected rats; **, P = 0.01 compared with PBS-injected rats.b PG-APS polymers (15 ,ug of rhamnose per g [body weight]) injected i.p. into five rats 36 days before sacrifice.' PBS injected i.p. into five rats 36 days before sacrifice.

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50 r

1-0

Io-

E

0

Ea)

45

40(

35 F

20 60 80

Days After Injection100 120

FIG. 4. Temporal relationship between delayed onset of anemiaand arthritis in a female Lewis rat injected i.p. with PG-APSpolymers (5 ,ug of rhamnose per g). This rat did not develop anemiaor arthritis until 42 and 50 days, respectively, after injection of a lowdose of PG-APS. The anemia was maximal at the onset of arthritis.

with bony proliferation and ankylosis, leading to persistentlyelevated joint scores because of ankle enlargement due tonew bone formation, but no gross evidence of active inflam-mation was detected by edema and erythema. Anemia wasbetter correlated with active joint inflammation than rela-tively inactive chronic joint destruction (Fig. 1) and usuallyimmediately preceded reactivation of arthritis in individualrats (Fig. 4). Similarly, increased spontaneous sedimentationof RBCs was associated with recurrence of anemia andarthritis (Table 5) and returned to normal values duringremission of active inflammation.

Rats studied during the first spontaneous reactivation ofarthritis, 15 days after PG-APS injection, developed severeanemia which was accompanied by significantly decreasedhemoglobin and RBC counts (P - 0.001, Table 6). The meancorpuscular volume of the PG-APS-injected rats was slightlylower than that for control rats. PG-APS-injected rats alsohad elevated WBC counts, which were closely correlatedwith HCT values (r = 0.93) and joint scores (r = 0.94). Thebone marrows of these rats had decreased numbers of RBCprecursors relative to WBC precursors, as demonstrated bytheir significantly elevated bone marrow myeloid precursors/erythroid precursor ratios (Table 6). Bone marrow histologyrevealed normal-to-increased cellularity, with increasednumbers of megakaryocytes and granulocyte precursors, inthe PG-APS-injected rats compared with PBS-injected con-trols.

DISCUSSION

Anemia associated with acute and chronic systemic in-flammation induced by PG-APS has several unique featureswhich make it a useful model to study the anemia of chronicinflammation. PG-APS-induced anemia was protracted, as itpersisted for at least 10 months after a single aqueous i.p.injection, and spontaneously relapsed and remitted, with itsclinical course correlating with exacerbations and remissionsof arthritis. Anemia following bacterial lipopolysaccharide(endotoxin) injection is transient and very mild (17). Forma-lin-killed Corynebacterium parvum injected into mice (27),infection with M. arthritides (11) or BCG (26), and footpadinjection of complete Freund adjuvant (25) induce anemiawhich is monophasic, with no evidence of spontaneousrecurrence, and is limited to at most 8-weeks duration (26).PG-APS is resistant to biodegradation (39) and persistswithin macrophages for at least 4 months after injection (8,35). Spontaneous reactivation of inflammation following asingle local or systemic injection of PG-APS is a character-istic feature of inflammation induced by these phlogisticcomplexes and may be due to slow intracellular degradationof polymers with episodic release from tissue stores (14).

In the anemia of chronic disease, serum iron, transferrin,and TIBC are low despite normal or increased bone marrowand tissue iron stores (13, 24, 48). Rats injected with PG-APShad significantly lower serum iron and transferrin concen-trations in the chronic phase of inflammation compared withthose of control rats, and there was a strong correlationbetween iron and transferrin levels and the activity ofarthritis. Macrophages within the liver, spleen, and mesen-teric lymph nodes had increased stainable iron, particularlywithin giant cells and granulomas. Fibrotic, more-advancedhepatic granulomas had more densely staining iron thanrecently formed, active granulomas with necrotic centers,suggesting increased sequestration of iron with chronicinflammation. Mice injected with Freund adjuvant haveslightly decreased plasma iron, but decreased TIBC andtransferrin saturation develop only late in the course ofanemia (25) and splenic iron is diminished, rather thanincreased, after repeated injections of adjuvant (28). Theanemia secondary to BCG infection is not associated withhypoferremia (26). Injection of C. parvum into rats leads toa prompt decrease in serum iron and retention of iron withinmononuclear phagocytes but an increased plasma TIBC (3).Similarly, mice with turpentine-induced inflammation (2) orinfected with M. arthritidis (11) have a prompt decrease inplasma iron but an increase in transferrin and TIBC. Thus,PG-APS-induced chronic anemia in rats more closely mimics

TABLE 5. HCTs, sedimentation rates, and joint scores during reactivation of PG-APS-induced inflammation

PG-APS-injected rats" PBS-injected ratsRat no.or mean HCT (%) Sedimentation Joint score HCT % Sedimentation Joint scorerateb ~~~~~~~~~~~~~~~~rate

1 36 15 8 47.5 0.5 02 47.5 0.5 0 48 0.5 03 28.5 10 8 49 1 04 31.5 24 4.5 48.5 0.5 05 30 36 7 48.5 0.5 0

Mean + SEM 34.7 ± 3.4' 17.1 + 6.1" 5.5 ± 1.5' 48.3 ± 0.3 0.6 ± 0.1 0

a Values measured 21 days after i.p. injection of PG-APS (15 ,ug of rhamnose per g [body weight]).b Spontaneous sedimentation of RBC measured as percentage of total capillary tube volume 30 min after collection.Pp < 0.003 compared with PBS-injected rats.

d p = 0.02 compared with PBS-injected rats.

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ANEMIA INDUCED BY BACTERIAL CELL WALLS 1183

TABLE 6. Blood and bone marrow parameters during the first reactivation of PG-APS-induced arthritis

Mean ± SEM"Materialinjected" HCT t Hemoglobin No. of RBC MCV' No. of WBC Bone marrow myeloid

(g/dl) (106/mm3) (tim3) (103/mm3) precursor/erythroidprecursor ratio

PG-APS 33.7 ± 2.9* 11.5 ± 0.9* 5.73 + 0.42* 58.7 ± 1.5** 38.5 ± 6.0* 3.4 ± 0.5*PBS 47.7 ± 0.7 16.2 ± 0.2 7.65 ± 0.12 62.4 ± 0.7 6.9 ± 0.7 0.8 ± 0.2

a Values measured 15 days after i.p. injection of PG-APS (15 ,ug of rhamnose per g [body weight]) or PBS (1 ml) during the first spontaneous reactivation ofarthritis.

b Values shown are the mean for five rats per group. *, P < 0.001 compared with PBS-injected rats; **, P = 0.03 compared with PBS-injected rats.' Mean corpuscular volume, calculated by HCT/RBC.

the clinical abnormalities of iron metabolism found in theanemia of chronic disease than previously described modelsof chronic inflammation or infection.The demonstration that the PG fraction induced anemia is

consistent with previous observations that polymeric PG isthe active moiety responsible for most inflammatory reac-tions induced by PG-APS fragments from a variety ofbacterial species (20, 41). Digestion of PG by the murami-dase, mutanolysin, abrogates acute and chronic arthritis (20)and prevents acute vascular permeability changes in thegut-associated lymphoid tissue induced by PG-APS (R. B.Sartor, unpublished data). Purified PG is readily degraded bylysozyme, in contrast to PG covalently bound to APS (1).This observation is consistent with the transient anemia andarthritis which resolved by 14 days after injection of isolatedPG with no evidence of recurrence, while PG-APS producedprotracted, spontaneously relapsing anemia and arthritis.Purified group A streptococcal APS produced transientperipheral edema, as previously described (4), but no anemiaor chronic arthritis.The mechanism of anemia induced by PG-APS is un-

known but probably involves both decreased RBC survivaland synthesis. The rapid decrease in HCT during the acutephase and spontaneous recurrence of anemia, combinedwith the slightly decreased RBC half-life measured by 5"Crlabeling, indicates that RBC survival is diminished. Therewas no evidence of gastrointestinal or external loss of RBCs,and the purified PG-APS preparation used for these studiesdid not contain streptococcal hemolysin. Patients with rheu-matoid arthritis have a 20% decrease in RBC survival (43),and rats with adjuvant arthritis have a circulating 51Cr-RBChalf-life of 10.2 days compared with 13.4 days in controls(28). One mechanism of shortened RBC survival is phago-cytosis of RBCs by reticuloendothelial cells, which occurs inC. parvum-treated mice (27). Splenic macrophages normallyclear senescent RBCs from the circtilation. Macrophages inthe spleen, liver, bone marrow, and lymph nodes can phago-cytose R13Cs that have subtle membrane damage or arecoated with antibody or complement (7). Activation ofmacrophages is a common feature of many types of inflam-mation and has been postulated to be important in thepathogenesis of the anemia of chronic disease (24, 33, 37).PG-APS activates macrophages in vitro and in vivo (39;Bristol and Schwab, submitted), and intravenous injection ofPG-APS leads to phagocytosis of RBCs by macrophageswithin mesenteric lymph nodes (34). PG activates comple-ment (12), and Pryzwansky et al. (30) have demonstrated invitro aggregation of RBCs and phagocytic cells via PG-APS-complement (C3b) bridging. Erythrophagocytosis byactivated reticuloendothelial cells could explain increasedstainable iron within macrophages of the liver, spleen, andmesenteric lymph nodes of PG-APS-injected rats with ane-mia. Another possible explanation of increased macrophage

iron is sequestration of lactoferrin-iron complexes afterliberation of lactoferrin from the secondary granules ofPG-APS-activated polymorphonuclear leukocytes.The primary defect in the anemia of chronic disease

appears to be the lack of an appropriate bone marrowresponse to the slight decrease in RBC survival (13, 48).Figure 2 shows only a slightly increased destruction of RBCsduring the relatively abrupt recurrence of anemia 3 to 5weeks after PG-APS injection, indicating variable suppres-sion of RBC synthesis during different phases of experimen-tal inflammation. Activated macrophages have been shownto suppress erythropoiesis both during stimulation with LPS(36, 37) or in disseminated histoplasmosis infection (50).Mouse peritoneal macrophages activated by human recom-binant tumor necrosis factor or Cryptococcus neoformansinfection suppress erythroid progenitor cell growth (33). Lee(24) speculated that IL-1 mediates the anemia of chronicdisease, but it is apparent that IL-1 has no direct erythroidinhibitory effect (21, 33, 37), although this monokine acti-vates polymorphonuclear leukocytes to release lactoferrin,causes a decrease in serum iron (24), and is produced byscattered bone marrow cells (44). IL-1 has indirect regula-tory effects on hematopoiesis by stimulating bone marrowstromal cells (15), T lymphocytes (19), fibroblasts (21), andendothelial cells (38) to release multilineage hematopoieticgrowth factors and activating fibroblasts to release solublefactors that regulate early hematopoietic progenitor cells(51). Thus, IL-1 may be responsible for the leukocytosisobserved after PG-APS injection. Recently, Tracey et al.(46) demonstrated that twice-daily administration of tumornecrosis factor to rats for 8 days induced a progressiveanemia which appeared within 3 days and was associatedwith a leukocytosis. We postulate that the chronic anemiainduced by PG-APS is a result of peptidoglycan activation ofmacrophages which liberate monokines, including tumornecrosis factor (45) and IL-1 (Bristol and Schwab, submit-ted), and soluble inflammatory mediators, such as prosta-glandin E2 (49), which may suppress erythropoiesis directlyor indirectly. Alternatively, stimulated T cells, which havebeen implicated in the pathogenesis of PG-APS-inducedinflammation (47), may suppress the bone marrow, as hasbeen demonstrated in rheumatoid arthritis (43) and BCGinfection in mice (29).PG-APS-induced inflammation in rats provides an excel-

lent model for examining protracted, spontaneously relaps-ing anemia with modestly decreased RBC survival andaltered iron metabolism associated with well-characterizedarthritis. PG-APS is chemically and antigenically defined andcan activate macrophages in vivo or in vitro, permittingmore thorough evaluation of mechanisms of bone marrowsuppression. Activation of macrophages by PG-PS may be acommon mechanism of the anemia accompanying chronicinfection by many bacterial species.

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1184 SARTOR ET AL.

ACKNOWLEDGMENTSWe thank Ann K. Schwab, Julia Blevins, Joseph J. White, and

Roger R. Brown for expert technical assistance; Shirley Willard fortyping the manuscript; and Eugene Orringer, Stephen A. Stimpson,and Kevin Connally for reviewing the manuscript.

This work was supported by Public Health Service grants AM01112, AR 25733 and DK 34987 from the National Institutes ofHealth and the National Foundation for Ileitis and Colitis.

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