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Antimicrobial Agents and Chemotherapy, July 2005, p. 3053-3055, Vol. 49, No. 7
0066-4804/05/$08.00+0     doi:10.1128/AAC.49.7.3053-3055.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Prevention of the Cryptic Epitope SLAYGLR within Osteopontin Does Not Influence Susceptibility to Candida albicans Infection

Ikuko Sato,1* Nobuchika Yamamoto,2 Harumi Yamazaki,2 Seiji Hashimoto,1 Motohiro Hino,1 Fumihiko Sakai,2 and Akihiko Fujie1

Fermentation Research Laboratories,1 Exploratory Research Laboratories, Fujisawa Pharmaceutical Co., Ltd., 5-2-3 Tokodai, Tsukuba, Ibaraki, 300-2698, Japan2

Received 25 August 2004/ Returned for modification 31 December 2004/ Accepted 27 March 2005


   ABSTRACT
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The effect of an antiosteopontin antibody (M5 Ab) reacting with the SLAYGLR sequence within osteopontin on the host susceptibility to infection was investigated in a murine model of disseminated candidiasis. The treatment with anti-tumor necrosis factor alpha antibody enhanced fungal infection, while the treatment with M5 Ab did not affect the infection.


   TEXT
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Biological tumor necrosis factor alpha (TNF-{alpha}) blockers such as infliximab and etanercept are well-known efficacious agents for the treatment of rheumatoid arthritis (RA). In spite of the effectiveness of these blockers, the accompanying increase in opportunistic infection has created much concern in the last several years (4, 13). Osteopontin (OPN), an extracellular matrix protein, has been suggested as a potential mediator, promoting joint destruction in patients with RA (2, 5, 9, 12). This factor functions as a cytokine, leading to type-1 immunity by promoting the production of gamma interferon (1). We previously reported that M5 Ab, an antiosteopontin antibody (Ab) reacting with the SLAYGLR sequence within OPN exposed by thrombin cleavage, suppresses the development of symptoms and the progress of RA in a murine model (14). In this study, the effects of M5 Ab and anti-TNF-{alpha} Ab on the host susceptibility to infection with pathogenic fungi were compared in a murine model of systemic infection.

Systemic mouse infection. Disseminated candidiasis was induced in 5-week-old, female ICR mice by intravenous inoculation via the lateral tail vein with 0.2 ml of a cell suspension of pathogenic Candida albicans. Abs were intravenously administered at 500 µg/mouse 1 day before and 6 days after infection. Cyclophosphamide (CP; Shionogi, Osaka, Japan) was administered intraperitoneally at 200 mg/kg of body weight 4 days before infection to induce a leukopenic condition in the mice. The polymorphonuclear leukocyte counts were less than 100/µl on day 6 after administration (3). The optimal inoculated number of C. albicans cells for the murine model of disseminated candidiasis was determined by monitoring the survival rates. Mice with moderate infectious conditions (6 x 105 CFU/mouse) were selected due to their good sensitivity to medication. Anti-TNF-{alpha} Ab (clone MP6-XT3; Pharmingen), anti-OPN Abs (IBL, Gunma, Japan), and the control immunoglobulin G (IgG) (rat IgG [Chemicon, Temecula, CA] and rabbit IgG [R&D Systems Inc., Minneapolis, MN]) were intravenously administered to the mice. Survival curves for each group are illustrated in Fig. 1. The infected, untreated mice and the infected, IgG-treated mice almost all died within 15 days. Figure 1A shows that all mice treated with CP died on the second day after infection. Death among the mice treated with anti-TNF-{alpha} Ab occurred significantly earlier than that in mice treated with IgG, as shown by a log rank test for comparing survival curves. The effects of anti-OPN Abs were also examined (Fig. 1B and C). Anti-N-terminal OPN Ab reacts to the N-terminal sequence of the mouse OPN, while anti-C-terminal OPN Ab reacts to the C-terminal sequence of the thrombin-cleaved, amino-terminal end of mouse OPN (8). Our results show that there is no significant difference in the effects of anti-OPN Ab and IgG on survival time (Fig. 1B and C). These observations strongly indicate that the administration of anti-TNF-{alpha} Ab caused a more severe infection than that experienced by the control group, and this effect was not observed with anti-OPN Abs.




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  		FIG. 1. Effect of antibodies in disseminated candidiasis. Eight mice were included in each group. (A) Effect of anti-TNF-{alpha} Ab and CP. {lozenge}, normal; {square}, rat IgG; {blacktriangleup}, anti-TNF-{alpha} Ab; •, CP. (B) Effect of M5 Ab. {lozenge}, normal; {square}, rabbit IgG; {blacktriangleup}, M5 Ab. (C) Effect of anti-OPN Ab. {lozenge}, normal; {square}, rabbit IgG; {blacktriangleup}, anti-C-terminal OPN; •, anti-N-terminal OPN. ***, results significantly different from those with the control Ab by log rank test (P < 0.001).

 
Target organ assay. A target organ assay was carried out to determine the degree of C. albicans dissemination in mouse kidneys at various times during infection. Antibodies were administered intravenously at 500 µg/mouse 1 day before infection. After the mice were sacrificed, their right and left kidneys were excised for assessment of Candida dissemination and for measurement of cytokine concentration, respectively. In the assessment of Candida dissemination, an aliquot of the kidney homogenate was plated on YMD (yeast extract-maltose extract-dextrose) agar plates as described previously (6). The numbers of CFU were counted after 24 h of incubation at 37°C and expressed as log CFU/kidney. The concentrations of protein and cytokines were determined using the Bio-Rad protein assay (Bio-Rad; catalog no. 500-006) and an enzyme-linked immunosorbent assay. Figure 2 shows the similarities of CFU changes in the M5 Ab-treated group and the IgG-treated group during infection. All mice treated with M5 Ab survived until the seventh day. On the other hand, the numbers of CFU in the anti-TNF-{alpha} Ab group showed a slight increase 1 day after infection. There was a statistically significant difference in the numbers of CFU between the anti-TNF-{alpha} Ab group and the IgG group on the third day. The time course study of CFU in the kidneys indicated that a count of 6.5 to 7.0 log CFU/kidney results in death (data not shown). Consistently, an increase in C. albicans CFU caused early death in mice treated with anti-TNF-{alpha} Ab.



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  		FIG. 2. Effect of anti-TNF-{alpha} Ab (A) and M5 Ab (B) on CFU in disseminated candidiasis. Four mice were included in each group. (A) Closed bars, normal; open bars, rat IgG; hatched bars, anti-TNF-{alpha} Ab. (B) Closed bars, normal; open bars, rabbit IgG; hatched bars, M5 Ab. Viable counts are given as mean log10 CFU ± 95% confidence interval/kidney. *, results significantly different from those with the control Ab by Dunnet's multiple-comparison test (P < 0.05).

 
In the present study, we found that infection with C. albicans was promoted by treatment with anti-TNF-{alpha} Ab. In mice treated with anti-TNF-{alpha} Ab, an increase in CFU and a high mortality rate were observed. Interestingly, the interleukin-1 and TNF-{alpha} levels in the kidneys were up-regulated in mice treated with anti-TNF-{alpha} Ab (data not shown). We considered that the TNF-{alpha} levels were increased by an unknown feedback mechanism as a result of the Ab treatment. The treatment with anti-TNF-{alpha} Ab initially suppressed innate immunity, followed by an induction of proinflammatory cytokines probably as a protection against pathogens, including C. albicans. On the other hand, in M5 Ab-treated mice, there was no significant regulation of the cytokine levels induced in response to C. albicans infection compared to that of the control mice. The increases in numbers of CFU and mortality in this group resembled those of the control group. These results led us to conclude that the inhibition of the cryptic epitope of OPN generated by thrombin digestion does not influence innate immunity. On the contrary, several studies have found that OPN plays an important role against infection with many pathogens (7, 10, 11). Koguchi et al. reported that production of interleukin-12 and gamma interferon caused by Mycobacterium bovis BCG infection was significantly suppressed by the 10A16 Ab, which reacts with the carbonyl moiety of thrombin-cleaved human OPN (7). We further investigated this subject to define the pivotal portion of OPN that provided protection from pathogens by using several anti-OPN Abs. We evaluated the effects of anti-C-terminal murine OPN Ab, which corresponds to 10A16 (8), and anti-N-terminal murine OPN Abs on C. albicans infection in mice. However, treatment with neither the anti-N-terminal OPN Ab nor the anti-C-terminal OPN Ab resulted in an increase in susceptibility to C. albicans infection. The use of a different model system can explain the reason for the difference between the results of Koguchi et al. and our results. In our in vivo infection model, the elimination of pathogens is dependent mainly on neutrophils; however, the in vitro model used by Koguchi and coworkers may be dependent on T lymphocytes. RA patients treated with infliximab or etanercept were observed to have an increased risk of infection with many pathogens (4). Our observations indicate that the innate immunity provided by neutrophils and monocytes is important in the protection from opportunistic infection in RA patients treated with TNF-{alpha} blockers. This study demonstrates that several epitopes within OPN may act independently to provide various immu-nological functions. In conclusion, we suggest that a neutralizing Ab that reacts with the cryptic epitope of SLAYGLR in mouse OPN may be efficacious in the treatment of RA patients without adverse events of serious infection.


   ACKNOWLEDGMENTS
 
We are grateful to Uma Devi M. Palanisamy and David Barrett for assistance in the preparation of the manuscript.


   FOOTNOTES
 
* Corresponding author. Mailing address: Fermentation Research Laboratories, Astellas Pharma Inc., 5-2-3 Tokodai, Tsukuba, Ibaraki, 300-2698, Japan. Phone: 81-29-847-8611. Fax: 81-29-847-8313. E-mail: ikuko.sato{at}jp.astellas.com. Back


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Antimicrobial Agents and Chemotherapy, July 2005, p. 3053-3055, Vol. 49, No. 7
0066-4804/05/$08.00+0     doi:10.1128/AAC.49.7.3053-3055.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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