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Previous Article | Next Article 
Infection and Immunity, March 2003, p. 1470-1480, Vol. 71, No. 3
0019-9567/03/$08.00+0 DOI: 10.1128/IAI.71.3.1470-1480.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.
and Scott J. Hultgren1*
Departments of Molecular Microbiology,1 Pediatrics,2 Pathology and Immunology,4 Internal Medicine, Washington University School of Medicine, Saint Louis, Missouri 63110,5 Pathology Department, University of Utah, Salt Lake City, Utah 841323
Received 12 September 2002/ Returned for modification 12 November 2002/ Accepted 9 December 2002
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ABSTRACT |
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B and p38 mitogen-activated protein kinase. Also, reverse transcription-PCR analysis demonstrated that bladder epithelial cells express CD14 mRNA. Thus, the molecular machinery utilized by bladder epithelial cells for the recognition of E. coli is very similar to that described for traditional innate immune cells, such as macrophages. In contrast, the A498 renal epithelial cell line did not express CD14, was hyporesponsive to LPS stimulation, and demonstrated poor IL-6 responses to E. coli. |
INTRODUCTION |
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The well-established in vitro and in vivo models for the interaction between Escherichia coli and uroepithelial cells make this system ideal for the investigation of pathogen recognition by epithelial cells. The ability of uropathogenic E. coli (UPEC) to establish infection in the urinary tract depends on its ability to express surface-adhesive organelles that facilitate colonization of the uroepithelium. P pili are produced by pyelonephritic strains of E. coli and mediate binding to globoseries glycolipids that predominate in the kidney (22). Consequently, P pili have been shown to be critical in the ability of E. coli to cause pyelonephritis (42). Type 1 pili bind to mannose-containing glycoproteins present on the bladder epithelial surface and are critical in the establishment of cystitis (31, 36). Type 1 pili are under an on-off phase variation control (6), and colonization of the bladder selects for type 1 piliated phase variants (11, 23).
Despite the distinct receptor specificities of P and type 1 pili, E. coli strains expressing either of these adhesive organelles have been demonstrated to augment bladder and kidney epithelial cell cytokine production compared to isogenic nonpiliated strains (14, 44). However, bacterial attachment mediated by these different adhesive pili results in the activation of distinct signaling pathways (16). The binding of P-piliated E. coli to globoside receptors present on kidney epithelial cells appears to activate interleukin-6 (IL-6) and IL-8 production via a predominantly lipopolysaccharide (LPS)-independent mechanism (7, 17). The role of Toll-like receptor 4 (TLR4) in this process is controversial. It has been reported that the LPS-independent activation of cytokine induction in kidney epithelial cells is TLR4 dependent (7, 17). In contrast, TLR4 has also been reported to be missing in kidney epithelial cells (3). The mechanism of activation of bladder epithelial cells by type 1 piliated E. coli is equally confusing. Several reports have argued that type 1 pili directly activate uroepithelial cytokine production and that LPS plays only a minor role (15, 47). These conclusions were based in large part on studies with A498 kidney cells. However, studies with bladder epithelial cells demonstrated that LPS is the primary bacterial factor activating cytokine production and that the role of type 1 pili is to augment the presentation of LPS to the LPS receptor complex on the bladder epithelial cells (3, 44). Thus, the discrepancies regarding the roles of type 1 pili and LPS in bladder cell activation may be explained by differences in the cell lines utilized for analysis.
LPS is the predominant component of the outer membrane of gram-negative bacteria, and its recognition by host cells requires an array of proteins. LPS-binding protein (LBP) and soluble CD14 (sCD14) are present in the serum and facilitate the transfer of LPS to membrane-bound CD14 (mCD14), a glycosylphosphatidylinositol-linked receptor on the surfaces of some host cells (24). It is thought that mCD14 subsequently interacts with TLR4, the signaling component of the LPS receptor (4). A secreted molecule known as MD-2 physically interacts with the extracellular domain of TLR4 and significantly enhances host cell responses to LPS (45, 51). TLR2 has also been reported to interact with certain lipid A structures, as well as with the lipoproteins that are intimately associated with LPS (18, 19). In host cells lacking expression of mCD14, sCD14 can partially compensate for the absence of this receptor under some circumstances (13, 32). Investigators using A498 cells as a model have reported that uroepithelial cells are CD14 negative (3, 15) and thus hyporesponsive to LPS stimulation.
The host signaling cascades that occur following TLR ligation involve a conserved cytoplasmic domain known as the Toll/IL-1 receptor (TIR) domain (38). The TIR domain of TLRs interacts with the adaptor proteins MyD88 and/or TIRAP, which subsequently recruits IL-1 receptor-associated kinases (IRAKs) to the receptor (21, 30, 50). Once an IRAK becomes activated, it dissociates from the receptor complex and leads to the activation of TRAF-6, an event that facilitates the downstream activation of I-B kinase (IKK) and p38 mitogen-activated protein (MAP) kinase via a Tak-dependent mechanism (46, 49). The result of these signaling events is the nuclear translocation and transactivation of NF-B, leading to the production of proinflammatory mediators and the up-regulation of costimulatory molecules (29). In addition, the activation of TLR2 has been shown to activate a phosphatidylinositol 3-kinase/Rac-1-dependent pathway, which leads to the transactivation of NF-B by Akt (1).
In this study, we sought to definitively assess the role of LPS and type 1 pili in the activation of bladder epithelial cells and to clarify discrepancies in the literature by analyzing multiple cell lines of uroepithelial origin. In contrast to the A498 cell line, all of the bladder epithelial cell lines tested were responsive to LPS. Furthermore, CD14 is expressed by bladder epithelial cells and, together with TLRs, mediates cellular activation following challenge with type 1 piliated E. coli or LPS.
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MATERIALS AND METHODS |
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Cell culture. 5637 epithelial cells (derived from a bladder carcinoma; ATCC HTB-9), T24 epithelial cells (derived from a bladder carcinoma; ATCC HTB-4), J82 epithelial cells (derived from a bladder carcinoma; ATCC HTB-1), and A498 epithelial cells (derived from a kidney carcinoma; ATCC CRL-7908) were cultured in RPMI medium (Gibco BRL, Carlsbad, Calif.) plus 10% fetal bovine serum (FBS; Sigma, St. Louis, Mo.) at 37°C in a water-saturated atmosphere containing 95% air and 5% CO2. THP-1 monocyte/macrophage cells (derived from a monocytic leukemia; ATCC TIB-202) were cultured in RPMI medium containing 10% FBS, 1.5 g of sodium bicarbonate/liter, 4.5 g of glucose/liter, 1 mM sodium pyruvate, and 0.05 mM 2-mercaptoethanol.
IL-6 stimulation assay.
5637, T24, J82, and A498 cells were seeded into 24-well plates at 0.3 x 105 to 1 x 105 cells per well and grown to confluence over 2 days. THP-1 cells were seeded into 24-well plates at 5 x 105 cells per well in a medium containing 50 mM phorbol 12-myristate 13-acetate (PMA) and allowed to differentiate for 2 days. Forty-eight-hour bacterial cultures were pelleted by centrifugation, resuspended in phosphate-buffered saline (PBS), and diluted to a concentration of 108 CFU/ml (except for the dose curve experiments, where multiple bacterial stock dilutions were generated). Ten microliters of the bacterial suspension was added to appropriate wells containing 1 ml of fresh medium to achieve a final bacterial concentration of 106 CFU/ml. Titers of bacterial suspensions were determined at the outset of each experiment to verify the number of live bacteria added to the wells. The bacteria were spun onto the cells by low-speed centrifugation and incubated at 37°C for 6 h. After the incubation, the supernatants were collected, cell debris and bacteria were removed by centrifugation, and samples were frozen at -80°C until they were assayed by using a human IL-6 sandwich enzyme-linked immunosorbent assay (R & D Systems, Minneapolis, Minn.). A similar protocol was used for determining the effects of IL-1
, tumor necrosis factor alpha (TNF-) (R & D Systems), LPS (Sigma), and the bacterial lipoprotein Pam3-Cys-Ser-Lys4-OH (Boehringer Mannheim, Indianapolis, Ind.) on cellular activation.
Signaling inhibition studies. 5637 cells were preincubated with 10 µM MG-132 or 5 µM SB203580 (both from Calbiochem, San Diego, Calif.) for 1 h, after which the cells were either left unstimulated or treated with the indicated stimulants. T24 cells were preincubated with 20 µM MG-132 or 50 µM SB203580 for 1 h, after which the cells were either left unstimulated or treated with the indicated stimulants. Supernatants were analyzed as described above.
Immunohistochemistry.
T24 cells were plated in 24-well plates on coverslips at 0.3 x 104 to 1 x 104 cells per well and were grown over 2 days. This lower cell density was utilized in order to better evaluate individual cells. After stimulation, cells were fixed with 3.7% paraformaldehyde-0.2% Triton X-100 in PBS for 10 min. After three 5-minute washes in PBS, coverslips were blocked with 1% bovine serum albumin and 0.2% milk in PBS (PBS-BB) for 15 min. Coverslips were then stained with 1 µg of anti-NF-B p65 (BD Pharmingen, San Diego, Calif.)/ml in PBS-BB overnight at 4°C. As a negative control, coverslips were stained without a primary antibody. After three 5-min PBS washes, coverslips were incubated with 3 µg of Cy3-conjugated F(ab')2 donkey anti-mouse immunoglobulin G (IgG) (Jackson Immunoresearch Laboratories, West Grove, Pa.)/ml for 1 h at room temperature.
CD14 inhibition. To block CD14-mediated responses, cells were preincubated with My4 (Beckman-Coulter, Fullerton, Calif.), a monoclonal anti-CD14 antibody, at 5 µg/ml or with the same concentration of a mouse IgG2b isotype control (Sigma) for 30 min at 37°C. The cells were subsequently either left unstimulated or treated with the indicated stimulants, and supernatants were analyzed as described above.
Flow cytometry. Cell suspensions were prepared from confluent cells growing as monolayers in T25 flasks and detached from the surface by using 0.02% EDTA in PBS. Between 2.5 x 105 and 5 x 105 cells were stained with My4 or the IgG2b isotype control at 5 µg/ml. Subsequently, the samples were incubated with biotinylated goat anti-mouse IgG (1:100) and finally phycoerythrin (PE)-conjugated streptavidin (1:600) (both from BD Pharmingen). Cells were analyzed by using a FACScalibur flow cytometer, and 12,000 live cells from each sample were collected. Data were analyzed by using Cell Quest software. The live cell population was defined by using propidium iodide staining.
CD14 mRNA reverse transcription-PCR (RT-PCR). Following the protocol of Funda et al. (8), total RNA was isolated from cultured cells with TRIzol (Gibco BRL) according to the package protocol. No additional efforts were made to remove genomic DNA. First-strand cDNA was prepared from 4 µl of total RNA product by using 200 U of Superscript II reverse transcriptase (Gibco BRL) in a reaction mixture containing 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol, 1.5 µM oligo(dT)12-18 primers, and 0.125 mM each deoxynucleotide triphosphate (dNTP) at 42°C for 50 min, then 70°C for 15 min. Three microliters of the cDNA product was subjected to PCR amplification with 2.5 U of Taq polymerase (Invitrogen, Carlsbad, Calif.) in a solution containing 10 mM Tris-HCl (pH 8.8), 50 mM KCl, 2 mM MgCl2, 0.2 mM each dNTP, and 0.8 mM each primer. Cycling parameters were as follows: 30 cycles of 94°C for 45 s, 57°C for 45 s, and 72°C for 2 min, followed by a 10-min final extension at 72°C. Primers for human CD14 (Invitrogen) were 5'-GCTGTGTAGGAAAGAAGCTA-3' (sense) and 5'-TTTAGAAACGGCTCTAGGTTG-3' (antisense). Since these primers span the single intron of the CD14 gene, expected product sizes are 356 bp from genomic DNA and 284 bp from mRNA (8). Primers for amplification of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Invitrogen) were 5'-TCATCTCTGCCCCCTCTGCT-3' (sense) and 5'-CGACGCCTGCTTCACCACCT-3' (antisense), with an expected product size of 440 bp (from either mRNA or genomic DNA). PCR products were run on 1.5% agarose gels stained with ethidium bromide and viewed with a UV transilluminator.
LPS tolerance. LPS tolerance was induced in 5637 and T24 bladder epithelial cells by incubating the cells with LPS from E. coli O55:B5 (Sigma) at 1 µg/ml for 16 to 20 h at 37°C. Following the incubation, cells were washed with PBS (plus Mg2+ and Ca2+), and fresh medium was added to the cells. Cells were subsequently either left unstimulated or treated with the indicated stimulants, and supernatants were analyzed as described above.
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RESULTS |
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B and p38 MAP kinase activation is required for bladder epithelial responses to LPS and E. coli.
The production of IL-6 following LPS stimulation generally involves the activation of NF-B and p38 MAP kinase; however, NF-B-independent pathways have been described (5). Stimulation of T24 bladder cells with type 1 piliated E. coli led to a redistribution of NF-B p65 from the cytoplasm to the nucleus, consistent with the activation of this transcription factor (Fig. 3a and b). Proteasome inhibitors have been shown to block NF-B translocation to the nucleus by preventing the degradation of IB (39), whereas p38 MAP kinase inhibitors appear to block the transactivation of NF-B in the nucleus (46). Preincubation of T24 bladder cells with the proteasome inhibitor MG-132 blocked NF-B p65 translocation and IL-6 production following challenge with FimH+ E. coli (Fig. 3c and e). Preincubation of bladder cells with the p38 MAP kinase inhibitor SB203580 blocked IL-6 production despite translocation of NF-B p65 to the nucleus (Fig. 3d and e). Similar results were observed when 5637 cells infected with FimH+ E. coli were pretreated with these inhibitors (Fig. 3f). As expected, both inhibitors blocked bladder cell IL-6 responses to IL-1 and LPS (data not shown). These results argue that the nuclear translocation of NF-B and the activation of p38 MAP kinase-dependent signal transduction cascades are required for the induction of bladder epithelial IL-6 following stimulation with inflammatory molecules and E. coli.
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, LPS (500 ng/ml), or FimH+ or FimH- E. coli. My4 had no effect on unstimulated or IL-1-stimulated cells but significantly reduced IL-6 production induced by LPS (65% inhibition), AAEC185/pUT2002 (FimH-) (89% inhibition), or AAEC185/pSH2 (FimH+) (77% inhibition) (Fig. 6). My4 had no effect on binding or invasion by AAEC185/pSH2 (data not shown).
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B and p38 MAP kinase. Therefore, TLRs would be predicted to be important signaling receptors in bladder epithelial cells, which have been shown to express tlr4 and tlr2 mRNA (3, 44). Bäckhed et al. transiently transfected a dominant-negative TLR4 construct into T24 cells and observed a modest decrease in LPS responsiveness (2). Prestimulation of macrophages with LPS renders them refractory to subsequent LPS stimulation, a phenomenon termed LPS tolerance. Though the precise molecular mechanism of LPS tolerance is unclear, it has been shown that LPS-tolerant macrophages have decreased TLR4 surface expression and also fail to activate IRAKs following LPS restimulation (34, 37). Cross-tolerance between IL-1 and LPS has been demonstrated, adding further support to the idea that LPS tolerance is the consequence of disrupted TIR domain-mediated signaling (33, 34). To further address the role of TLR signaling in bladder epithelial responses, we investigated the ability of 5637 bladder cells to become tolerant to LPS stimulation.
To induce tolerance, bladder epithelial cells were incubated with LPS for 16 to 20 h, after which time the cells were washed and either left unstimulated or stimulated with IL-1, TNF-, LPS, or bacterial lipoprotein (LPP). 5637 cells had defective responses to IL-1 (60% inhibition), LPS (87% inhibition), and LPP (94% inhibition) following tolerance induction (Fig. 7a). However, the responses to TNF- were unaffected in tolerant 5637 cells. These results demonstrate that LPS tolerance can be induced in bladder epithelial cells and argue that LPS-tolerant 5637 cells have a defect in the MyD88/IRAK pathway, as this signaling pathway is shared by IL-1R and TLRs but not by TNF receptors.
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DISCUSSION |
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In this study, 5637 (bladder), T24 (bladder) and J82 (bladder) cells were all shown to respond to LPS, whereas A498 cells (kidney) failed to respond to even the maximal dose of LPS tested. The ability to recognize LPS directly correlated with the production of IL-6 following challenge with both FimH+ and FimH- E. coli. Specifically, the threshold of activation for the LPS-hyporesponsive A498 cells by E. coli was 1,000 times and 10 times higher than it was for T24 and 5637 bladder cells, respectively. This is consistent with previous reports arguing that T24 and 5637 cells recognize and respond to LPS on the surfaces of type 1 piliated E. coli cells (3, 44). At a stimulatory dose of 106 CFU/ml, FimH-expressing E. coli enhanced IL-6 production from the uroepithelial cell lines over production with the isogenic FimH- mutant strain. This augmentation has been shown previously to be a consequence of type 1 pilus-mediated bacterial invasion (44). However, FimH- bacteria still activate a significant IL-6 response in 5637, T24, and A498 cells, indicating that functional (adhesive) type 1 pili are not required to induce such a response. It is not known which bacterial product(s) is recognized by A498 cells; however, the present data would suggest that LPS may not mediate the activation of these cells (7). It is possible that the lack of a functional LPS receptor complex in A498 kidney cells unmasks the stimulatory capacity of other bacterial molecules, as has been demonstrated using a lipid A mutant strain of Neisseria meningitidis (25, 41).
The present data support the model that bladder epithelial cell lines are LPS responsive and that the ability to recognize LPS is a critical event in the induction of innate responses against FimH+ and FimH- E. coli. In macrophages, the primary host cell receptor complex for LPS consists of mCD14 and TLR4/MD-2, which lead to the activation of p38 MAP kinase and the nuclear translocation of the transcription factor NF-B (35, 38). It has been proposed that epithelial cells recognize pathogens through pathways that are fundamentally different from those in macrophages (9, 40). Moreover, if one were to assume (based on examination of the A498 cells alone) that uroepithelial cells do not express mCD14 or utilize sCD14 for anti-E. coli responses, then an alternate pathway for LPS recognition would have to exist (9, 17, 47).
To define the LPS receptor complex utilized by bladder epithelial cells, the signal transduction cascades leading to IL-6 production were investigated. Stimulation of bladder epithelial cells with E. coli led to the nuclear translocation of NF-B p65. In addition, preincubation of bladder epithelial cells with the proteasome inhibitor MG-132 (which blocks NF-B translocation) or with the p38 MAP kinase inhibitor SB203580 completely blocked the induction of IL-6 following bacterial challenge. These findings demonstrate a role for these signal transduction cascades in the activation of bladder epithelial cells by LPS on type 1 piliated E. coli.
To further characterize the receptors involved in the generation of LPS-mediated responses, the expression and role of CD14 were evaluated. The responses of 5637, T24, and J82 bladder epithelial cell lines to LPS were inhibitable with the monoclonal anti-CD14 blocking antibody My4, demonstrating that CD14 is a component of the LPS receptor complex on all of these bladder cell lines. It was also shown that bladder epithelial responses to FimH+ and FimH- E. coli require CD14. Cell surface expression of CD14 was shown by using flow cytometry with an anti-CD14 antibody for 5637, T24, and J82 bladder cells; however, consistent with previous results, A498 cells did not express CD14 (17). RT-PCR analysis confirmed the presence of CD14 mRNA in all three bladder epithelial cell lines. In contrast, Bäckhed et al. were unable to demonstrate amplification of CD14 mRNA from T24 cells (2). The reason for this discrepancy is unclear, but it may result from growth conditions or cell passage number, as CD14 expression on human nonmyeloid cells in vitro has been shown to decrease with time in culture (26). Since our functional experiments were performed in the presence of serum, a role for sCD14 in contributing to the activation of these mCD14-expressing cells cannot be excluded, although no CD14 was detected on the surfaces of A498 kidney cells. In addition, our bladder cell cultures for flow cytometry and RT-PCR contained no LPS, and sCD14 has been shown to require the presence of LPS in order to bind to the surface of CD14-negative nonmyeloid cells (48). Thus, our data argue that at least a portion of the surface CD14 on bladder cells is likely to be endogenously produced.
TLRs are pathogen pattern recognition receptors that play a critical role in innate immunity. TLR4 and TLR2 have been implicated in LPS recognition by macrophages, but other cell types also express these receptors, including 5637 and T24 bladder epithelial cells (3, 44). LPS tolerance is a phenomenon first described for macrophages where following a prestimulation with low to intermediate concentrations of LPS, cells become refractory to a second LPS stimulation. The current understanding of LPS tolerance would argue that the primary mechanism underlying the nonresponsiveness of tolerant cells is the disruption of signal transduction cascades initiated by TIR domain-containing receptors, potentially superimposed on a loss of TLR4 expression on the cell surface (37). Bladder epithelial cells that were prestimulated with LPS had significantly reduced responses to IL-1, LPS, and LPP but had normal responses to TNF-. These results demonstrate that LPS tolerance can be induced in uroepithelial cells. Furthermore, the observation that the response to TNF- is unaffected in LPS-tolerant cells indicates that the cells are not globally suppressed but instead have a specific signaling defect that maps to TIR domain-containing receptors (such as TLRs) or early downstream signaling molecules. When LPS-tolerant bladder cells were stimulated with FimH+ or FimH- E. coli, IL-6 production by these cells was significantly inhibited. These data argue that bladder epithelial activation following bacterial stimulation is mediated by TLR activation, consistent with the findings of Bäckhed et al. (2).
Since signaling via multiple TLRs is likely rendered nonfunctional in the LPS-tolerant state, it is not possible to conclusively state which member(s) of this receptor family mediates E. coli recognition on bladder epithelial cells. However, the importance of LPS for bladder epithelial cytokine responses, the inability of C3H/HeJ mice (TLR4 mutant) to effectively clear E. coli-induced bladder infections, and the effect of the dominant-negative TLR4 mutant demonstrated by Bäckhed et al. (2) suggest that TLR4 is critical in E. coli recognition (20, 44). TLR2 has also been implicated in the recognition of LPS and its associated lipoproteins, suggesting a potential role for this receptor as well (19). To more closely mimic the LPS that would be encountered by host cells infected with whole bacteria, the LPS used in this study was not repurified to eliminate the trace lipoproteins that are intimately associated with commercially purified LPS preparations (18). Thus, it is possible that TLR2 activation is contributing to the LPS-driven stimulation we observed in bladder epithelial cells, particularly at higher doses of LPS. Ongoing studies aim to determine the respective roles of TLR4 and TLR2 in bladder epithelial responses to LPS and E. coli.
Epithelial cells are uniquely positioned to coordinate early host defenses against invading pathogens. However, in order to study the contribution of epithelial activation to the innate immune response, the molecules utilized by these cells to recognize pathogens must be defined. Significant progress has been made in understanding these details by using in vitro uroepithelial cell systems. However, this study further illustrates the fact that cell cultures are an imperfect representation of the in vivo epithelium and that it is often difficult to determine which, if any, of the cell lines provides the best representation of the in vivo epithelial surface. In this analysis, we utilized three independent bladder epithelial cell lines to gain a more comprehensive understanding of bladder epithelial responses to LPS and E. coli. The data from this investigation demonstrated that all of these bladder cell lines are LPS responsive and that this response is facilitated by the activation of NF-B and p38 MAP kinase through a CD14-TLR complex. The present data are consistent with those of Bäckhed et al. showing a role for TLR4 in LPS responsiveness in bladder epithelial cells. Also, the significantly impaired ability of C3H/HeJ mice to clear an experimental bladder infection (12, 20, 44) and a recent report that the murine bladder mucosa expresses numerous inflammatory genes following LPS challenge in vivo (43) are consistent with our findings. Thus, we argue that the bladder mucosal surface is primed to recognize and respond to LPS. In contrast, the A498 cell line is hyporesponsive to LPS and substantially less efficient at producing anti-E. coli responses than the bladder cell lines. Based on these results, we suggest that the bladder epithelium should be considered a CD14-expressing, LPS-responsive entity that is not accurately depicted by the A498 cell line. Alternatively, published results with the A498 cell line may in fact argue that there are fundamental differences in innate responses of bladder and kidney epithelia, as has been proposed by Bäckhed et al. (3). Future studies using in vivo analysis and primary cell lines will help to further characterize CD14-TLR interactions and bladder epithelial responses to UPEC during an acute urinary tract infection. The methods employed here may be applied to other epithelial cell types to determine whether the CD14-TLR receptor complex is employed by these epithelia to recognize and respond to LPS.
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ACKNOWLEDGMENTS |
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We thank M. Chapman for helpful discussions throughout the generation of this paper.
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FOOTNOTES |
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Present address: Department of Pathology, University of Alabama at Birmingham, Birmingham, AL 35294.
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