INTRODUCTION

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Six to ten percent of women suffer from recurrent, uncomplicated urinary tract infections (UTIs) caused predominantly by Escherichia coli (15, 29). Host characteristics of this unusually susceptible population are not completely defined, but it has been noted that women with recurrent UTIs have diminished antibody responses to some E. coli antigens (13). Whereas the immunological basis of this observation is not completely understood, hyporesponsiveness may be due to the absence of T helper subsets required for induction of anti-E. coli B-cell responses.

Bacterial superantigens are known to stimulate T-cell populations through a series of events that begins with their binding to major histocompatibility complex (MHC) class II molecules outside the conventional antigen binding site. Superantigens are then presented by class II molecules to a specific subset of T cells, causing mass proliferation followed by apoptosis or anergy of that T-cell subset. Staphylococcal enterotoxin B (SEB) specifically stimulates the V3⁺, V12⁺, V14⁺, V15⁺, V17⁺, and V20⁺ T-cell subsets in humans (17) and the V8⁺ T-cell subset in mice (22). The effects of SEB on the immune system begin with the secretion of cytokines at 0 to 24 h, leading to the clonal expansion of V8⁺ T cells (in mice) from 1 to 3 days followed by deletion and anergy within 3 days (3, 4, 9, 16, 24, 35). We investigated the effect of SEB treatment in vivo on the resolution of an induced E. coli UTI and observed that SEB given prior to inoculation with E. coli did not affect subsequent UTI resolution, whereas SEB administered concurrently with the inoculum enhanced UTI resolution. Further studies on SEB-treated mice revealed increased production of cytokines in the spleen and bladder and of interleukin-6 (IL-6) in the liver.

	MATERIALS AND METHODS

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Mice. Female, 8- to 10-week-old BALB/c mice were purchased from Harlan Sprague-Dawley, Indianapolis, Ind.

UTI induction. Mice were infected, as described previously (12), with the clinically isolated uropathogenic E. coli strain 1677 (34). Briefly, a catheter was inserted into the bladder of an anesthetized mouse, and a dose of 10⁸ 1677 CFU in phosphate-buffered saline was delivered in a 20-µl volume. The intensity of infection was determined by dilution plating bladder homogenates on Levine's EMB agar (Difco) and calculating the log₁₀ (CFU/milligram of bladder).

SEB administration. Fifty micrograms of SEB (Sigma) was administered intraperitoneally (i.p.) in 100 µl of RPMI 1640 medium (Mediatech, Fisher Scientific) (36) at either 3 or 7 days prior to UTI induction or concurrently with the bladder inoculation. The purity of the SEB was verified by the manufacturer by sodium dodecyl sulfate-gel electrophoresis and was reported to have 31% protein and less than 0.2% SEA in a certificate of analysis.

Toxicity testing. Strain 1677 was prepared the same as for UTI induction, and 10⁸ cells were cultured with various amounts of SEB (0 to 50 µg) in a total volume of 120 µl at 37°C for 30 or 60 min. The cell suspension was dilution plated on EMB agar to calculate the log₁₀ (CFU/milliliter).

Measurement of urinary GAG and pH. Urine was collected from SEB-treated and untreated mice (10 mice per group) at 2, 4, and 6 h after i.p. injection by gently pressing on the lower abdomen and was pooled for each group. Glycosaminoglycan (GAG) levels were assayed as described previously (33) and were normalized to creatinine levels. pH levels were obtained by pipetting urine onto pH indicator paper (Whatman).

Detection of cytokine mRNA expression by reverse transcription-PCR (RT-PCR). Spleens, bladders, and livers were removed at 6, 12, and 24 h from animals in different treatment groups, immediately snap-frozen in liquid nitrogen, and stored at 70°C. Total RNA was prepared by using the Ultraspec RNA isolation system (Biotecx, Houston, Tex.) according to the manufacturer's instructions, with the addition of QIAshredders (Qiagen) after cell lysis to remove cellular debris. The quality of RNA was confirmed by spectrophotometry (A₂₆₀/A₂₈₀) and agarose gel electrophoresis. In a total volume of 30 µl, 1.5 µg of RNA was reverse transcribed with 2.5 µM oligo(dT) primer (Research Genetics, Huntsville, Ala.)-1 mM deoxynucleoside triphosphate (Promega)-450 U of Moloney murine leukemia virus reverse transcriptase (Promega) at 37°C for 60 min followed by 95°C for 5 min. Equal amounts of cDNA from identical treatment groups were pooled. Cytokine detection PCR primer sequences for IL-1, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12 p40, granulocyte-macrophage colony-stimulating factor (GM-CSF), gamma interferon (IFN-), transforming growth factor  (TGF-), tumor necrosis factor alpha (TNF-), and -actin were published previously (8) and synthesized by Research Genetics. With the -actin primers as an internal control, each reaction mixture contained 0.25 µM each -actin and cytokine primers, 1.5 µl of cDNA, 200 µM deoxynucleoside triphosphates, 2 mM MgCl₂, and 2.5 U of AmpliTaq DNA polymerase (Perkin-Elmer Cetus) and was amplified for 24 to 30 cycles of 45 s at 95°C, 1.5 min at 60°C, and 1.5 min at 72°C, followed by a final extension for 7 min at 72°C. Negative controls with omission of cDNA were used. PCR products were separated on a 2% agarose gel and visualized by staining with SYBR Green I (FMC, Rockland, Maine). Photographs were taken with a DC40 Kodak digital science camera and analyzed with Electrophoresis Documentation Analysis software (Kodak, Rochester, N.Y.).

Statistical analysis. Infection resolution data were analyzed by analysis of covariance on rank transformed data (30).

	RESULTS

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Effects of SEB treatment on resolution of induced UTIs. When animals were pretreated with SEB 3 or 7 days before infection induction, the expanded population of V8⁺ T cells presumably had undergone apoptosis or become unresponsive (3, 22, 35). Hypothetically, with an incomplete T-cell repertoire, E. coli may be able to establish a more intense bladder infection. SEB pretreatment at 3 or 7 days before inoculation had no effect on bladder colonization compared to control mice (Table 1). However, when SEB was given at the time of inoculation, the infection progressed in a host with proliferating T cells and abnormally high cytokine levels. In this environment, the UTI resolved more rapidly than in non-SEB-treated mice (Fig. 1). Mice given SEB had significantly lower numbers of E. coli in the bladder at 6 h, 1 day, 2 days, and 3 days (P = 0.01, 0.0001, 0.001, and 0.0001, respectively).

View larger version (16K): [in this window] [in a new window]   FIG. 1.   SEB injection given concurrently with UTI induction accelerates infection resolution. Mice were given 50 µg of SEB i.p. () at the time of intravesical inoculation with 108 CFU of strain 1677. Control animals () received the bacterial inoculum only. A total of 8 to 24 mice were assayed per time point when repeat experiments were pooled.

Physiological effects of SEB. While it is most likely that the observed effect of SEB on UTI resolution was mediated by stimulated T cells or their products, or other MHC class II⁺ cells, it is possible that SEB is bactericidal or causes other physiological effects that could lead to decreased E. coli colonization of the bladder. To rule out direct toxicity of SEB for E. coli, we cultured strain 1677 with fivefold dilutions of SEB from 50 to 0 µg and measured bacterial viability after 30- and 60-min incubations. For all dilutions of SEB tested, the mean log₁₀ (CFU/ml) ± standard error of the mean (SEM) were 8.86 ± 0.04 and 7.65 ± 0.10, compared to 8.91 and 7.76 in the control cultures lacking SEB, after the 30- and 60-min incubations, respectively. Therefore, we concluded that SEB had no significant bactericidal effect on strain 1677.

Cytokine mRNA expression following SEB administration. In the absence of physiological evidence for SEB-enhanced UTI resolution, we investigated possible immunological effects. When SEB and E. coli inoculum were given at the same time, an initial decrease in the level of bladder colonization occurred within 6 h, and the greatest reduction in bacterial numbers was seen in the first 24 h (Fig. 1). Because cytokines induced by SEB could be responsible for this early effect, cytokine profiles were obtained from spleen cells to examine cytokine regulation of trafficking immune cells, and cells in the bladder, to determine local cytokine production at the site of infection. All cytokine mRNA levels were considered relative to the -actin transcript (Fig. 2 and 3, upper band in each lane). The results for the 6-, 12-, and 24-h time points did not differ in either the spleen or bladder (data not shown). Spleen cells of SEB-treated mice displayed an mRNA cytokine profile typical of cells stimulated by SEB (Fig. 2) (9, 32). IL-2 and IL-4 expression was upregulated significantly, and IL-6 and IL-10 levels were slightly increased. IL-1, GM-CSF, IFN-, TGF-, and TNF- were produced constitutively. IL-4 was the only cytokine detected above control levels in animals receiving an infection only. IL-5 and IL-12 were not induced in any treatment group (data not shown).

View larger version (29K): [in this window] [in a new window]   FIG. 2.   Cytokine mRNA expression levels in spleen cells following SEB treatment in vivo and/or induced UTI. Lane 1, infection only; lane 2, SEB plus infection; lane 3, SEB only; lane 4, untreated control. The upper band in each lane is the -actin transcript. Results from the 6-, 12-, and 24-h time points did not significantly differ; therefore, a representative gel from each group is shown.

View larger version (56K): [in this window] [in a new window]   FIG. 3.   Cytokine mRNA expression levels in bladder cells following SEB treatment in vivo and/or induced UTI. Lane 1, infection only; lane 2, SEB plus infection; lane 3, SEB only; lane 4, untreated control. The upper band in each lane is the -actin transcript. Results from the 6-, 12-, and 24-h time points did not significantly differ; therefore, a representative gel from each group is shown.

View larger version (60K): [in this window] [in a new window]   FIG. 4.   IL-6 mRNA expression levels in liver cells 12 h after SEB treatment in vivo. Lanes 1 to 4, RT-PCR products from four individual mice receiving 50 µg of SEB i.p.; lanes 5 to 8, RT-PCR products from four untreated control mice. The upper band is the -actin transcript.

	DISCUSSION

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The administration of SEB in vivo has allowed us to examine several aspects of host response to UTIs. SEB belongs to a family of superantigens, secreted by certain bacteria such as staphylococci, that are among the body's natural flora. We originally postulated that a woman with recurrent UTIs may have had asymptomatic staphylococcal infections during which SEB was secreted, and exposure to the superantigen created an unresponsive population of T cells purportedly necessary for the resolution of UTIs. Our experiments examining this hypothesis demonstrated that pretreatment with SEB did not affect the course of UTI resolution as postulated, indicating that the V8⁺ T-cell subset may not play a major role in host defense against UTIs. To be more conclusive, we would need to confirm that the SEB treatment conditions used here and by others (3, 22, 35) led to depletion of V8⁺ T cells. In contrast to pretreatment, when SEB and E. coli infection were given concurrently, there was an approximate 90% decrease in the level of bladder colonization at 6 h and 1 to 3 days after infection in SEB-treated mice compared to untreated controls. This finding would be consistent with an SEB-induced release of stored cytokines within 6 h, followed by de novo cytokine synthesis. It is worth mentioning that the animals injected with SEB may also be receiving an amount of SEA less than 0.1 µg. Future experiments will involve testing low doses of purified SEA in our system to reveal any role it may have in UTI resolution.

After possible physiological effects of SEB (bactericidal effects, lowered urinary pH, or GAG increase) were discounted, we examined components of early host defense that could be stimulated by SEB treatment to accelerate resolution of UTIs. In this regard, it is conceivable that enhanced clearance of bacteria in the bladder could be initiated by cytokines produced either by spleen cells or locally by cells in bladder tissue. We first determined the types of cytokines produced in the spleen that could be transported to the bladder. Spleen cells from SEB-treated mice produced primarily IL-2 and IL-4 and to a lesser extent IL-6 and IL-10. Potential effects of IL-2 and IL-4 include stimulation of T and B cells and differentiation and growth of many cell types in the spleen (14). IL-6 and IL-10 expression may lead to proliferation of numerous cell types and enhance B-cell proliferation, respectively (14). All four of these cytokines can be made by splenic T and B cells, in addition to many other cell types (14, 25). Our results are in agreement with a previous report on cytokines stimulated by SEB treatment in vivo in which IL-2, IL-4, IL-10, and IFN- were detected in splenic CD4⁺ T cells within 2 to 48 h by RT-PCR (9, 32). Similar results were seen by others in lymph node cells and serum (11, 20). Because IFN- message was also present our untreated controls, we were unable to determine if IFN- transcription was upregulated by SEB.

SEB can also produce local effects in the bladder, since it can migrate through all organs in a few hours (24). To evaluate this, we looked for increased cytokine mRNA expression in bladder cells following i.p. injection of SEB. IL-1, IL-6, GM-CSF, and TNF- mRNA were expressed above control levels in the bladder. The bladder has numerous cell types that are able to secrete these cytokines upon stimulation. These include epithelial cells (14), macrophages (14, 25), mast cells (25), dendritic cells (14), neutrophils (5), natural killer (NK) cells (25), and T cells (21, 25). Furthermore, several studies have shown that SEB stimulates not only V8⁺ T cells but also many other effector cells bearing MHC II receptors belonging to the nonadaptive immune response repertoire, including T cells (27) and NK cells. In vitro experiments revealed that after 24 h, SEB directly stimulated cytokine secretion (6) and killer activity of NK cells, which had enhanced antibacterial activity against E. coli strains including a pyelonephrogenic strain (10). The cytokines that we detected belong to a family that act collectively to enhance the inflammatory response, recruit effector cells, and resist infection (18). For example, IL-1 and TNF- were shown to have nonspecific anti-Pseudomonas activity in a granulocytopenic mouse model (1), and IFN-, TNF-, and IL-6 were protective in mycobacterial infections (2). Mast cells were shown to release stored TNF to help confine bacterial infections (7), modulate neutrophil influx, and enhance bacterial clearance in murine acute septic peritonitis (23). Other researchers have shown that urinary IL-6 levels increased within 2 h after exposure to E. coli and that E. coli can stimulate epithelial cells to initiate release of neutrophil chemoattractants (31).

We also found induction of IL-6 message in liver cells from mice treated with SEB, suggesting that production of acute-phase proteins may have been stimulated and thus could have played a role in accelerating infection resolution (14).

Our data of cytokine mRNA profiles suggest a cascade of SEB-stimulated immunological events leading to enhanced bacterial clearance from the bladder. First, we detected mRNA for a group of inflammation-enhancing cytokines in the bladder (IL-1, IL-6, GM-CSF, and TNF-), and this family of cytokines, along with SEB-stimulated effector cells (possibly NK or T cells) could be responsible for the early (6 h) reduction of E. coli in the bladder. Second, the production of IL-2, IL-4, IL-6, and IL-10, inferred from transcripts in the spleen, may contribute to infection resolution at later time points (1 to 3 days). Conceivably, these cytokines would activate T and B cells in the spleen, which could then enter the circulation, travel to the bladder, and enhance bacterial clearance. Third, IL-6 mRNA detected in liver cells of SEB-treated mice may stimulate secretion of acute-phase proteins, which could travel in the serum to the bladder, bind bacteria, and promote bacterial phagocytosis through opsonization (28). Whereas this proposed model needs to be verified, the current studies on SEB administration in vivo have allowed us to begin identifying some early host defense mechanisms necessary to resolve UTIs.