Immunology 1998 93 80–85

The dual role of interferon-c in experimental Staphylococcus aureus septicaemiaversus arthritis

Y.-X. ZHAO, I.-M. NILSSON & A. TARKOWSKI Departments of Rheumatology and Clinical Immunology, University ofGoteborg; Goteborg, Sweden

SUMMARY

To evaluate the role of interferon-c (IFN-c) in Staphylococcus aureus infection, we investigatedthe effects of supplementation with and neutralization of IFN-c during septicaemia and arthritisin a murine model. In vivo administration of IFN-c both before and after bacterial inoculationsignificantly decreased mortality on one hand but enhanced the development of arthritis on theother. Treatment of mice with anti-IFN-c monoclonal antibodies (mAb) before and after bacterialinoculation did not significantly influence the survival rate but decreased the frequency andseverity of arthritis. The beneficial effect of supplementation with IFN-c on septicaemia wascorrelated to the increased phagocytosis and bacterial clearance from liver and kidneys. Thedown-regulation of the development of arthritis by anti-IFN-c mAb was accompanied by thedecreased serum tumour necrosis factor-a, interleukin-6 and interleukin-1b levels. These resultsdemonstrate a significant role for IFN-c in simultaneous protection against septicaemia butpromotion for the development of septic arthritis.

INTRODUCTION Salmonella typhimurium.10,11 In contrast, IFN-c synergizeswith TNF to mediate symptoms of Gram-negative shock.12,13

Staphylococcus aureus, a Gram-positive extracellular-growingIn addition, IFN-c has been shown to have variable effects in

bacterium, remains a common pathogen in serious infections.autoimmune arthritis. For example, systemically administered

Pathogenic staphylococci are capable of causing a variety ofIFN-c protects mice from collagen type II-induced arthritis,

disease syndromes in humans ranging from a localized arthritiswhereas local administration of IFN-c promotes the develop-

to disseminated infection resulting in death. Host responsive-ment of arthritis.14,15 To explore the role of IFN-c in

ness to the infectious process induced by staphylococci isstaphylococcal infection, in the present study we investigated

poorly understood, although studies involving animal modelsthe effects of supplementation with and neutralization of

and human infections have implicated that cytokines inducedIFN-c on the development of septicaemia and arthritis both

by staphylococci and their products may play a central rolebefore and after inoculation with S. aureus. The results

in the disease pathogenesis.1 Production of tumour necrosisdemonstrate a significant role of IFN-c in protection against

factor-a (TNF-a), interleukin-6 (IL-6), IL-1b and interferon-csepticaemia but simultaneous promotion of the development

(IFN-c) has been found to increase in response to S. aureusof septic arthritis.

infection in vitro and in vivo.2–4 However, the direct role ofthese cytokines in the development of S. aureus infection,

MATERIALS AND METHODSincluding septicaemia and arthritis, has not been established.IFN-c, produced primarily by T cells and natural killer Mice

(NK) cells, has been shown to be a pleiotropic cytokine Male NMRI mice 8–10 weeks old, purchased from B&Kand plays an important role in many immunological Universal AB (Sollentuna, Sweden), were used. Each animaland inflammatory processes. It is known that IFN-c acti- weighed 27–31 g and was given food and water ad libitum.vates macrophages to kill intracellular parasites, includingListeria monocytogenes,5–7 Mycobacterium tuberculosis,8,9 and Bacteria and inoculation procedure

The S. aureus LS-1 strain used in the experiments has beenReceived 14 August 1997; revised 8 October 1997; accepted previously described.16 This bacterial strain was proved to be

9 October 1997. catalase- and coagulase-positive and produces large amountsAbbreviations: IFN-c, interferon-c; S. aureus, Staphylococcus of toxic shock syndrome toxin-1 (TSST-1).17 The bacteria

aureus. were cultured on blood agar for 24 hr and then reincubatedon blood agar for another 24 hr. A bacterial solution wasCorrespondence: Dr Y.-X. Zhao, The Toronto Hospital Arthritisprepared in phosphate-buffered saline (PBS). Mice were inocu-Centre, The Toronto Hospital, 13-415 Mc, 399 Bathurst Street,

Toronto, Ont., Canada M5T 2S8. lated intravenously (in the tail ) with 1×107 colony forming

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Role of IFN-c in murine S. aureus infection 81

units (CFU )/mouse. Viable counts were used to check the mice and then transferred to blood agar plates. After incu-bation for 24 hr, colonies were counted and presence ofamount of bacteria injected.S. aureus was confirmed by the presence of catalase andcoagulase activity. The results were expressed as the numberIFN-c treatment

Recombinant IFN-c (rIFN-c) was produced in vitro by Chinese of CFU per whole organ.19hamster ovary (CHO) cells transfected with murine IFN-c

Phagocytosisgene (kindly provided by Dr Morris, Department of BiologicalFor determination of phagocytic activity, uptake of fluoresceinSciences, University of Warwick, Coventry, UK). The super-isothiocyanate (FITC )-labelled bacteria by granulocytes andnatant containing rIFN-c was dialysed against PBS and furthermonocytes in peripheral blood was examined using a test kitconcentrated by polyethylene glycol 3000. Mice were injected(Phagotest, Orpegen Pharma, Heidelberg, Germany). In brief,intraperitoneally (i.p.) with 3×104 U of rIFN-c 2 hr beforefreshly drawn heparinized whole blood was incubated withor 3 days after the bacterial inoculation. The control micethe S. aureus LS-1 strain labelled with FITC20 for 10 min atreceived PBS at the same time as the IFN-c. The biological37°. The samples were then placed on ice to stop phagocytosis.activity of rIFN-c has been confirmed by the increase ofThe samples were treated with quenching solution for sup-phagocytic activity in naive mice injected with rIFN-c, aspressing fluorescence of the bacteria attached to the outsidedetermined by flow cytometry (see Results section).of the cells. The percentage of monocytes and granulocytesshowing phagocytosis (ingestion of one or more bacteria perAnti-IFN-c antibody treatmentcell ) and the phagocytic activity (fluorescence intensity perMice were injected i.p. with a single dose of 1 mg of IgG-ratcell ) was determined by flow cytometry (Becton Dickinson).anti-mouse IFN-c antibodies ( XMG1.2) (kindly provided by

Dr R. L. Coffman, Department of Immunology, DNAXAssessment of cytokine levels in seraResearch Institute, Palo Alto, CA) either 2 hr before or 3 daysThe cytokine levels in sera were determined by enzyme-linkedafter staphylococcal inoculation. One milligram of IgG-ratimmunosorbent assay (ELISA) or bioassay. TNF-a was quant-anti-ovalbumin antibodies was injected as a control substanceified using an ELISA system as described previously.21 IL-1baccording to the same protocol. The hybridoma producinglevels were determined using an IL-1b ELISA kit from R&DIgG-rat anti-ovalbumin antibodies was kindly provided by Drsystems (Minneapolis, MN ). The assay was performed asTelemo (Department of Clinical Immunology, Goteborgrecommended by the manufacturer. IL-6 levels were measuredUniversity, Sweden). The hybridoma supernatants were pre-by a bioassay using the cell clone B13.29, subclone B9 ascipitated with 50% of saturated ammonium sulphate andpreviously described.17dialysed against PBS. The biological activity of anti-IFN-c

monoclonal antibodies (mAb) has been confirmed by down-Statistical analysisregulation of in vivo delayed type hypersensitivity to oxazoloneThe differences between non-parametric values in the twoand in vitro nitrate (an NO− metabolite) production by antreatment groups were tested for significance by use of theendothelioma cell line in response to TNF and IFN-c (resultsMann–Whitney test. The differences between groups withnot shown).respect to the occurrence of mortality and arthritis wereanalysed by use of the x2 test. The data are expressed asClinical evaluation of mortality and arthritismean±SEM.All the mice were coded and were individually evaluated for

development of mortality and arthritis by two blindedobservers. The development of clinical arthritis was monitored RESULTSat regular intervals (3, 7 and 10 days after inoculation).

Effect of IFN-c administration on mortality and arthritisArthritis was defined as visible joint swelling or erythema ofat least one joint. The frequency of arthritis was expressed as Age- and weight-matched male NMRI mice were randomly

assigned to three experimental groups. All the mice received apercentage of the survivors which develop arthritis in eachgroup at each time-point. To evaluate the intensity of arthritis, single intravenous injection with 1×107 S. aureus LS-1. Group

1 and group 2 were injected i.p. with 3×104 U of rIFN-c pera clinical scoring (arthritic index) was carried out by using asystem where macroscopic inspection yielded a score of 0–3 mouse 2 hr before or 3 days after bacterial inoculation, respect-

ively. Group 3 received PBS. As shown in Fig. 1, IFN-cpoints for each limb (1 point=mild swelling and/or erythema;2 points=moderate swelling and erythema; 3 points=marked administration caused a striking decrease of mortality but a

simultaneous significant enhancement of arthritis. The signifi-swelling, erythema, and occasionally ankylosis). The arthriticindex was constructed by adding the individual scores of each cant difference with respect to mortality between the

IFN-c (–2 hr) group and the PBS group was seen from day 7limb, as previously described.18to day 14 after bacterial inoculation (P<0·05) (Fig. 1a). Thus,1 week after inoculation of S. aureus 50% of mice in theDetermination of bacterial growth

Growth of staphylococci in liver and kidneys was determined PBS group were dead compared with 8% of mice in theIFN-c (−2 hr) group. At day 10 after bacterial inoculation,by colony enumeration. The liver and kidneys were aseptically

removed and homogenized in 10 ml of medium. Appropriate 11 of 12 (92%) of mice in the PBS group were dead whereasonly 20–30% of mice in both IFN-c-treated groups succumbed.dilutions were made, and 0·1 ml of tissue suspensions or

heparinized blood was incubated on blood agar plates. For The mortality of the mice in the IFN-c (−2 hr) group, wassomewhat lower than that of the IFN-c (+3 days) group.bacteriological examination of joints, the isolates were

obtained with the help of charcoaled sticks from the joint of With respect to the arthritis (Fig. 1b, c), IFN-c (−2 hr) group

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Table 1. Growth of S. aureus in parenchymatous organs in micetreated with IFN-c before and after bacterial inoculation*

IFN-c IFN-c(−2 hr) (+3 days) PBS

Liver 4·1±3·2×104† 0·7±0·3×104‡ 26·8±14·3×104Kidneys 9·8±3·1×107 5·5±1·9×107 12·5±3·2×107Joint 5/5 (100%) 6/6 (100%) 7/8 (88%)

*Samples were aseptically obtained at day 5 after intravenousinoculation with 1×107 S. aureus LS-1 per mouse. Group IFN-c(−2 hr) received i.p. 3×104 U rIFN-c 2 hr before and group IFN-c(+3 days) received the same dose of rIFN-c 3 days after bacterialinoculation. Control group received PBS.

†Data are expressed as mean CFU±SE from at least five miceper group.

‡P<0·05 in comparison with PBS group.

found with respect to the bacterial isolation from jointsbetween the study groups.

The phagocytic activity of neutrophils and macrophages(Table 2) was determined by flow cytometry. The IFN-c(−2 hr) group displayed a significantly increased percentageof neutrophils being able to phagocytize S. aureus LS-1 strainas compared with the PBS group. In addition, the phagocyticactivity of leucocytes was tested in the non-infected micetreated with IFN-c for 24 hr. The percentage of phagocytesand the amount of ingested bacteria per cell in the IFN-c-treated group compared with the PBS group were 35·6±2·9versus 19·5±1·5 and 1564±213 versus 989±335, respectively.

The serum TNF-a levels in IFN-c-treated groups werehigher compared with those in the PBS group but no significantdifferences were found between the groups (data not shown).Similarly, there were no significant differences regarding theIL-6 and IL-1b levels between the groups (data not shown).

Effect of neutralization of IFN-c on mortality and arthritis

To assess the role of endogenous IFN-c production on thecourse of S. aureus infection, NMRI mice were treated with1 mg of rat anti-mouse IFN-c mAb given at 2 hr before or 3Figure 1. Effect of administration of rIFN-c on mortality (a) anddays after S. aureus inoculation. Control mice received 1 mgarthritis (b, c) in mice inoculated with S aureus. NMRI mice were

injected intravenously with 1×107 S. aureus LS-1. Group IFN-c of IgG-rat anti-ovalbumin antibodies according to the same(−2 hr) received i.p. 3×104 U rIFN-c 2 hr before and group IFN-c protocol. There was no difference with respect to the mortality(+3 days) received the same dose of rIFN-c 3 days after bacterialinoculation. Control group received PBS. Each group contained 12 Table 2. Phagocytic activity of granulocytes and macrophages (inmice; (a) mortality, (b) frequency of arthritis, and (c) severity of percentage) in mice treated with IFN-c before and after inoculationarthritis. with S. aureus*

IFN-c IFN-cdeveloped a more severe arthritis at day 3 (P<0·05) and both (−2 hr) (+3 days) PBSIFN-c-treated groups displayed higher frequencies of arthritis

Granulocytes (%) 44·4±5·0† 31·8±6·6 27·5±2·8at day 7 (P<0·05) after bacterial inoculation than the PBSFluorescence intensity 1265±183 963±70 1085±58group.Macrophages (%) 27·2±4·6 26·6±4·8 24·2±3·4The growth of bacteria in parenchymatous organs, phago-Fluorescence intensity 664±160 752±273 586±76cytic activity of leucocytes and cytokine levels in sera were

examined 5 days after bacterial inoculation. The bacterial*Samples were obtained from at least five mice per group at 5

counts in liver and kidneys (Table 1) were decreased in both days after intravenous inoculation with 1×107 S. aureus LS-1 perIFN-c-treated groups compared with the PBS group. Notably, mouse. Group IFN-c (−2 hr) received i.p. 3×104 U rIFN-c 2 hr beforethe number of staphylococcal colonies cultured from liver in and group IFN-c (+3 days) received the same dose of rIFN-c 3 daysthe IFN-c (+3 days) group were significantly lower than those after bacterial inoculation. Control group received PBS.

†P<0·01 in comparison with PBS group.in the PBS group (P<0·05). No significant difference was

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Role of IFN-c in murine S. aureus infection 83

between the two groups. Thus, 60% of mice in anti-IFNc (2 hr) versus 861±19 significantly decreased (P<0·05) comparedwith the control group at day 3 after bacterial inoculation. Ingroup developed mortality compared with 50% in the control

group; Forty per cent of mice in anti-IFN-c (+3 days) group anti-IFN-c (+ 3 days) group, The percentages of neutrophils(36±4 versus 44±2) and macrophages (29±6 versus 45±2)and the control group were dead, respectively. In contrast,

mice treated with anti-IFN-c mAb displayed decreased fre- decreased compared with the control group at day 6 afterbacterial inoculation. There was no difference regarding thequency and severity of arthritis compared with the controls

(Fig. 2). Thus, anti-IFN-c (−2 hr) group displayed at day 3 phagocytic activity of leucocytes at day 10 between the groups(data not shown).post-bacterial inoculation lower frequency (50% versus 88%)

and less severe arthritis (0·7±0·3 versus 2·6±0·6 compared Cytokine levels in sera, including TNF-a, IL-6 and IL-1b(Table 3), decreased generally in anti-IFN-c mAb-treatedwith the immunoglobulin-treated group. At day 7 the fre-

quency of arthritis was 63% in anti-IFN-c (−2 hr) group groups compared with the control group. Notably, IL-6 levelat day 10 significantly decreased in anti-IFN-c (+3 days)compared with 100% in the control group (P<0·05). In anti-

IFN-c (+3 days) group, both frequency (50% versus 83%) group compared with immunoglobulin group. UndetectableTNF-a and IL-1b levels were revealed in anti-IFN-c mAb-and arthritic index (0·2±0·2 versus 2·0±0·7) were significantly

higher (P<0·01) at day 10 after bacterial inoculation compared treated groups at day 10 after bacterial inoculation.with the control group.

Growth of bacteria in liver, kidneys and joints was exam-ined at days 3, 6 and 10 after bacterial inoculation. The

DISCUSSIONnumber of CFU in kidneys of anti-IFN-c (−2 hr) group(1·7±0·3×107) was significantly higher (P<0·05) than that In the present study, we investigated the effects of IFN-c

administration and neutralization, respectively, on murine S.in the control group (5·2±3·2×106 at day 3 after bacterialinoculation. No significant differences of bacterial growth in aureus septicaemia and arthritis. In vivo administration of

IFN-c both before or after bacterial inoculation increasedliver and joints were found between the groups (data notshown). survival on the one hand but enhanced the development of

arthritis on the other. Treatment of mice with anti-IFN-c mAbPhagocytic activity of leucocytes in mice treated withanti-IFN-c mAb decreased compared with the control group. before or after bacterial inoculation did not significantly

influence the survival rate but decreased the frequencies andIn anti-IFN-c (−2 hr) group, the load of bacteria per neutro-phil (944±82 versus 1172±49) and per macrophage (665±67 severity of arthritis. These results demonstrate a significant

Figure 2. Effect of neutralization of IFN-c on arthritis in mice inoculated with S aureus LS-1. NMRI mice were injectedintravenously with 1×107 S. aureus LS-1. Group anti-IFN-c (−2 hr) received i.p. 1 mg of IgG-rat anti-mouse IFN-c antibodies2 hr before and group anti-IFN-c (+3 days) received the same dose of the mAb 3 days after bacterial inoculation. The controlgroup received IgG-rat anti-ovalbumin antibodies according to the same protocol. Each group contained 10 mice. (a) Frequencyof arthritis in group anti-IFN-c (−2 hr), (b) severity of arthritis in group anti-IFN-c (−2 hr), (c) frequency of arthritis in groupanti-IFN-c (+3 days), and (d) severity of arthritis in group anti-IFN-c (+3 days).

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Y.-X. Zhao et al.84

Table 3. Serum cytokine levels in mice treated with anti-IFN-c mAb arthritis, such as collagen type II-induced arthritis, adjuvantbefore and after inoculation with S. aureus* arthritis and streptococcal cell wall-induced arthritis.14,24,25 In

the present study, in contrast to the decreased mortality,Anti-IFN-c Anti-IFN-c IFN-c-treated mice developed more frequent and more severe

(−2 hr) (+3 days) Control arthritis than the control group. Moreover, neutralization ofendogenously produced IFN-c led to a decrease of arthritis.

IL-1b (pg/ml ) Thus IFN-c may exert its proinflammatory effect locally inday 56±56 ND† 14±14 the affected tissues. In this respect, IFN-c has been implicated

3to promote arthritis in murine Borrelia burgdorferi infection.26day ND 305±185 209±56There was no clear-cut difference with respect to bacterial6isolates from the joints, suggesting that the development ofday 65±33 <7±7 338±140arthritis was not stringently correlated to the growth of10bacteria in the joints. Thus, the bacteria may just be a triggerIL-6 (pg/ml )of arthritic process. Alternatively, our non-quantitative wayday 2568±810 ND 3425±1596to determine bacterial growth in the joints was not precise3

day ND 1515±170 1800±100 enough to show more subtle differences.6 IFN-c can modulate production of other cytokine, such as

day 883±587 308±220‡ 1615±111 TNF-a, IL-1 and IL-6.27,28 These cytokines have been sug-10 gested to play a proinflammatory role in the bacterial infection1TNF-a (pg/ml ) and autoimmune arthritis.29,30 The exact role of these cytokines

day <31±31 ND 196±125 in S. aureus infection regarding the septicaemia and arthritis3 is not clear. In our experiments, there was no significant

day ND 209±209 2758±1175 difference in the levels of serum TNF-a, IL-6 and IL-1b in6

animals treated with IFN-c compared with those in controls.day <31±31 52±52 517±237However, blockade of endogenous IFN-c decreased the levels10of the TNF-a, IL-6 and IL-1b at later stages of the infection.These data could suggest that the IFN-c-mediated triggering*Samples were obtained from at least three mice per group afterof the proinflammatory cytokines was not directly involved inintravenous inoculation with 1×107 S. aureus LS-1 per mouse. Groupthe septicaemia but rather correlated to the development ofanti-IFN-c (−2 hr) received i.p. 1 mg of IgG-rat anti-mouse IFN-carthritis. TNF-a, IL-1b and IL-6 have all been suggested toantibodies 2 hr before and group anti-IFN-c (+3 days) received thebe critical cytokines mediating the pathology of the jointsame dose of the mAb 3 days after bacterial inoculation. Control

group received IgG-rat anti-ovalbumin antibodies. inflammation in autoimmune arthritis.29,30 Thus, it is reason-†ND, not done. able to assume that the protection afforded by anti-IFN-c mAb‡P<0·01 in comparison with the immunoglobulin control group. was, at least partly, due to a direct suppressive effect on

TNF-a, IL-1b and IL-6 secretion.Our findings provide evidence of the multifactorialrole for IFN-c in protection against septicaemia but promotion

character of the regulatory role of IFN-c in S. aureus infection.for septic arthritis.The differential role of IFN-c in staphylococcal septicaemiaIFN-c is a highly pleiotropic cytokine exerting manycompared with arthritis suggests that IFN-c can exert bothbiological activities on defence against bacterial infections. Itbeneficial and harmful properties. Our data suggest that earlycan activate macrophages and neutrophils to enhanceadministration of IFN-c can be used successfully to decreasemicrobicidal activity.22 However, IFN-c is also known to bethe mortality during the S. aureus septicaemia. However, thea potent inducer of major histocompatibility complex (MHC)proinflammatory property of IFN-c in local sites of infectionclass II antigens on various cells, and thus may promoteshould be considered.immune-mediated diseases.22 Therefore, IFN-c could poten-

tially play both a beneficial as well as a harmful role inbacterial infections. In the present study, IFN-c administration ACKNOWLEDGMENTSprotected mice from death, indicating that IFN-c is crucial

We thank Dr Coffman for providing the hybridoma which producesfor host defence in S. aureus sepsis. This beneficial effectIgG-rat anti-mouse IFN-c antibodies ( XMG1.2) and Dr Morris for

correlated well with an increased phagocytic activity of leuco- providing Chinese hamster ovary cells transfected with the murinecytes and efficient bacterial clearance. Our results are consistent IFN-c gene. We thank Mrs Lena Svensson and Mrs Margaretawith the recent study where rIFN-c administration followed Verdrengh for excellent technical assistance. This work was supported

by grants from the Goteborg Medical Society, the Swedish Associationby inoculation with S. aureus resulted in an increased elimin-against Rheumatism, the King Gustaf V 80 years Foundation, theation of bacteria from the spleen, liver and peritoneal cavityNanna Svartz Foundation, the Swedish Medical Research Council,of mice.23 In the present study, treatment of mice with rIFN-cthe Swedish Agency for Research Co-operation with Developing2 hr before bacterial inoculation led to even better outcomeCountries (SAREC), the University of Goteborg, the Tornspiran

compared with the mice treated with rIFN-c 3 days after Foundation, the A.-G. Crafoord Foundation, and the Gustaf Dahlenbacterial inoculation. This result indicates that in the early Foundation.stage of the infection IFN-c plays a beneficial role in resistanceto S. aureus sepsis.

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