Mast cell regulation of inflammation and gene expression during antigen-induced bladder inflammation in mice

RICARDO SABAN1, MARCIA R. SABAN1, NGOC-BICH NGUYEN1, TIMOTHY G. HAMMOND2 and BARRY K. WERSHIL3

1 Department of Physiology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104
2 Tulane Environmental Astrobiology Center, and Nephrology Section, Tulane University Medical Center, and Veterans Affairs Medical Center, New Orleans, Louisiana 70112
3 Departments of Pediatrics, Microbiology and Immunology, State University of New York Downstate Medical Center, Brooklyn, New York 11203-2098


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Mast cell numbers are significantly increased in bladder disorders including malignancy and interstitial cystitis, but their precise role has been difficult to determine. We characterized the role of mast cells on gene regulation associated with antigen-induced bladder inflammation in mice. For this purpose, we examined the responses in mast cell-deficient (KitW/KitW-v), congenic normal (+/+), and KitW/KitW-v mice that were reconstituted with bone marrow stem cells (BMR) to restore mast cells. All mice were actively sensitized and challenged intravesically with either saline or specific antigen. Bladder inflammation occurred in +/+ and BMR but not the KitW/KitW-v mice. Gene expression was determined using mouse cDNA expression arrays. Self-organizing maps, performed without preconditions, indicated gene expression changes dependent on the presence of mast cells. These genes were upregulated in bladders isolated from antigen challenge of +/+, not altered in KitW/KitW-v, and were upregulated in BMR mice. Taken together these results demonstrate an important role for mast cells in allergic cystitis and indicate that mast cells can alter their environment by regulating tissue gene expression.

self-organizing maps; gene array; mast cell-deficient mice; inflammation; mast cells; and interstitial cystitis


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
MAST CELLS ARE NORMALLY DISTRIBUTED throughout mucosal surfaces and connective tissues. They lie adjacent to epithelial cells, blood, or lymphatic vessels and near or within peripheral nerves (16). This distribution facilitates the exposure of mast cells to stimuli that can lead to activation, such as blood-borne antigens or neuropeptides. Furthermore, it places the mast cell at the interface of potentially critical host-pathogen interactions at mucosal surfaces. In turn, the mast cells’ distribution makes their cell products available to a variety of cell types in the microenvironment, including fibroblasts and other cells of the connective tissue, surface, or glandular epithelial cells, nerves, vascular endothelial cells, and genitourinary smooth muscle cells.

Although a central role of mast cells and basophils has been emphasized in acute immunologic reactions, it is now clear that these cells can also participate in more persistent and even chronic inflammatory or immunologic responses. For example, mast cells have been implicated in interstitial cystitis (IC), a chronic form of inflammation in the urinary bladder. IC affects millions of women and is characterized by severe pain, increased frequency of micturition, and even disability (18). The trigger in IC is not entirely known and pain often appears out of proportion to standard laboratory and pathological evaluation, suggesting a role for sensory nerves in its pathogenesis (13). Fundamental work regarding the participation of sensory nerves and mast cells in cystitis provided indirect evidence, such as increased numbers of mast cells in the detrusor and submucosa, and morphological evidence of mast cell activation and degranulation (4, 5, 22, 26). In addition, the extensive tissue remodeling seen in IC (20) along with increased urinary levels of histamine and tryptase (21) suggests a role for mast cells.

Numerous aspects of mast cell biology are influenced by the tissue microenvironment. For example, mast cell phenotype (mucosa or connective tissue type mast cell, as defined by the types of proteases expressed) is regulated the microenvironment (8). As a result, changes in the microenvironment produced by disease processes or immunologic responses may affect the phenotype expressed by various mast cell populations (9). Several factors have been identified that influence mast cell growth and function including interleukin-3 (IL-3), -4, -9, -10, nerve growth factor, and tissue factors such as stem cell factor (SCF) (7). Although these factors affect mast cell growth and development, it is also likely that mast cells alter and shape the nature of the tissue microenvironment.

In a previous study, we found that mast cells modulate experimental cystitis induced by intravesical instillation of substance P or lipopolysaccharide (LPS) in mice (1). We therefore hypothesized that the inflammatory response to antigen stimulation in the bladder requires mast cell participation and that mast cells would influence the tissue gene expression during the response. To examine this issue, we utilized an animal model of antigen-induced cystitis established in our laboratory (2, 12, 24) and confirmed by others (17, 19). The contribution of mast cells in the reaction was determined by examining the response in mast cell-deficient KitW/KitW-v, normal congenic (+/+) mice, and KitW/KitW-v mice that had their mast cell deficiency repaired by bone marrow reconstitution. This approach has been used to precisely define the role of mast cells in biological responses in vivo (see Ref. 32 for a review). Bladder inflammation was induced by intravesical challenge of sensitized mice with specific antigen, and gene regulation was determined by cDNA arrays. Self-organizing map (SOM) techniques (25) identified functionally gene clusters that depend on the presence of tissue mast cells for their expression, providing a powerful technical basis for future analysis of mechanisms of bladder inflammation.


    METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

Animals.
All animal experimentation described here was performed in conformity with the "Guiding Principles for Research Involving Animals and Human Beings" (OUHSC Animal Care and Use Committee protocol 00-109).

Three groups of 8-wk old female mice were used in these experiments. Genetically mast cell-deficient WBB6F1-KitW/KitW-v and congenic normal WBB6F1 (+/+) mice were purchased from Jackson Laboratories. Bone marrow-reconstituted (BMR) KitW/KitW-v mice were generated as previously described (30, 31; see below). Animals were maintained in animal housing facilities with filter hooded cages and allowed food and water ad libitum.

Bone marrow reconstitution.
Mutations at the W locus have profound effects on a variety of cell lineages, including mast cells, and bone marrow transplantation repairs the mast cell deficiency of KitW/KitW-v mice (30). Briefly, femoral and tibial bone marrow cells were harvested in Dulbecco’s modified Eagle’s medium (DMEM) from WBB6F1-+/+ mice. The cells were washed, and each KitW/KitW-v mouse was injected with 2 x 107 bone marrow cells in 0.2 ml of DMEM intravenously. Ten weeks later, the hematocrit was determined (to confirm that the mice had their anemia repaired), and these mice were then used for experiments. At the time of death, tissue was taken for histological examination to confirm the presence or absence of mast cells.

Antigen sensitization protocol.
All mice in this study were sensitized with 1 µg dinitrophenyl-4-human serum albumin (DNP4-HSA) in 1 mg alum on days 0, 7, 14, and 21, intraperitoneally. In normal mice, this protocol induces sustained levels of IgE antibodies up to 56 days postsensitization (11), and mast cell-deficient KitW/KitW-v mice have been shown to develop normal IgE responses in similar active immunization protocols (5). One week after the last sensitization, cystitis was induced.

Induction of cystitis.
Sensitized mice were anesthetized (40 mg/kg ketamine and 2.5 mg/kg xylazine ip) and then transurethrally catheterized (24 gauge, 3/4 in.; Angiocath, Becton-Dickson, Sandy, UT). Application of slight digital pressure to the lower abdomen drained the urine. The urinary bladders were instilled with either 150 µl of saline or antigen [DNP4-ovalbumin (DNP4-OVA); 1 µg/ml] infused at a slow rate to avoid trauma and vesicoureteral reflux (23). To ensure consistent contact of substances with the bladder, infusion was repeated twice within a 30-min interval, and the syringe was kept on the catheter. The latter ensured that there was no reflux or leakage for at least 30 min. Twelve mice were used per group. After instillation with saline (n = 12) or antigen (n = 12), mice were randomly distributed into the following groups: 1) RNA extraction (n = 3), 2) replicate of RNA extraction (n = 3), 3) morphological analysis (n = 6).

Morphological analysis.
The urinary bladder was evaluated for the infiltration of inflammatory cells and the presence of interstitial edema. A semi-quantitative score using defined criteria of inflammation severity was used to evaluate cystitis (24). A cross section of bladder wall was fixed in formalin, dehydrated in graded alcohol and xylene, embedded in paraffin, and cut serially into four 5-µm sections (8 µm apart). One set of slides was stained with hematoxylin and eosin and the other with Giemsa stain. Histology slides were scanned using a Nikon digital camera (model DXM1200) mounted on a Nikon microscope (Eclipse model E600). Image analysis was performed using a MetaMorph Imaging System (Universal Imaging, West Chester, PA). The severity of lesions in the urinary bladder was graded as follows: 1+, mild (infiltration of a low number of neutrophils in the lamina propria, and little or no interstitial edema); 2+, moderate (infiltration of moderate numbers of neutrophils in the lamina propria, and moderate interstitial edema); 3+, severe (diffuse infiltration of moderate to large numbers of neutrophils in the lamina propria and severe interstitial edema).

Sample preparation for cDNA expression arrays.
Three bladders from each group were homogenized together in Ultraspec RNA solution for isolation and purification of total RNA. Mouse bladders were pooled to ensure sufficient RNA for gene array analysis without amplification. RNA was DNase treated according to the manufacture’s instructions, and the quality of 10 µg RNA was evaluated by denaturing formaldehyde/agarose gel electrophoresis. This procedure was replicated using additional three bladders per each experimental group. Therefore, two pools of RNA were generated per experimental group for a total of six mice.

Mouse cDNA expression arrays.
cDNA expression arrays were determined as described before (23). Briefly, cDNA probes prepared from DNase-treated RNAs obtained from each of the experimental groups were hybridized simultaneously to four membranes containing Atlas Mouse cDNA Expression Arrays (Atlas no. 7741-1; Clontech Laboratories, Palo Alto, CA; for a complete list of genes present in this array, see http://www.clontech.com/atlas/genelists/index.html). Briefly, 5 µg of DNase-treated RNA was labeled with [{alpha}-32P]dATP and reverse-transcribed to cDNA, according to the manufacture’s protocol (Clontech Laboratories). The radioactively labeled complex cDNA probes were hybridized overnight to cDNA expression arrays (Clontech Laboratories) using ExpressHyb hybridization solution with continuous agitation at 68°C. After two high-stringency washes, the hybridized membranes were exposed to a phosphor-imaging screen overnight (Cyclone Storage System; Packard BioScience, Downers Grove, IL). The membranes were also exposed to X-ray film at -80°C with an intensifying screen for various lengths of time to determine the optimal exposure to generate equally intense hybridization signals for the housekeeping genes. Exposed X-ray film was scanned with Color OneScanner (Apple Computer, Cupertino, CA) to Adobe Photoshop software. Two different cDNA pools were hybridized to Clontech membranes, each representing the homogenate of three individual mouse bladders with one cDNA expression array.

Quantification.
The phosphor-imaging screen contains phosphor crystals that absorbs the energy emitted by the radioactivity of the sample and reemit that energy as a blue light when excited by a red laser. Results are presented as digital light units (DLU) and were interpreted by using OptiQuant image analysis software (Packard BioScience). Quantification of each detectable band was performed by measuring the DLUs generated using OptiQuant. To standardize the array, the background was subtracted, and within each membrane expression was calculated as percentage of ß-actin, with the expression values derived from multiple experiments utilized for cluster analysis. We set the following arbitrary criteria for determining which genes were dependent on the presence of tissue mast cells: the gene should be upregulated at least twofold by antigen challenge, the same gene should not be upregulated in the absence of mast cells, and finally, upregulation should be brought back by bone marrow reconstitution. A cutoff expression of 2% of ß-actin was used to avoid a null denominator when calculating the ratio of gene expression between antigen- and saline-treated tissues. Two different cDNA pools were hybridized to a cDNA expression array, each representing the RNA extracted from three individual mouse bladders for a total of six mice per group. The results for individual pools were averaged for analysis by Venn diagram analysis (see below).

Cluster analysis.
Genes presenting a similar time-dependent peak expression were identified in the mouse bladder experiments based on a Euclidean distance metric, after eliminating genes that failed to vary in expression level within an experiment by a factor of 3, and normalizing within experiments to a mean of 0 and a standard deviation of 1. Cluster analysis was performed using data normalized as a percentage of ß-actin. SOMs were used as described by Tamayo et al. (25). SOM is an unsupervised network learning algorithm which has been successfully used for the analysis and organization of large data files. We applied SOM to analyze the time course of gene regulation during LPS-induced cystitis (23), and others have shown that SOM is an excellent tool for the analysis and visualization of gene expression profiles (14, 25, 27). SOMs are a type of mathematical cluster analysis that is particularly well suited for recognizing and classifying features in complex, multidimensional data (25). The method has been implemented in the GeneCluster software, which performs the analytical calculations and provides easy data visualization (http://www.genome.wi.mit.edu). This focuses attention on the "shape" of expression patterns rather than on absolute levels of expression. The advantage of this approach is that large data sets can be clustered much faster than by using hierarchical clustering, because a lower number of clusters are assigned. Using GeneCluster software, SOMs were constructed by choosing a 6 x 4 grid that generated 24 clusters. As this type of analysis assumes that the data can be divided into a certain number of clusters and that they are well separated, it was also our concern to present the fewest clusters possible that would still give a clear picture of antigen-induced gene expression. Increasing the number of clusters by increasing the grid did not give us any additional correlation between genes. Venn Diagram and scatter plots of the same data were obtained using GeneSpring software (Silicon Genetics, Redwood City, CA). Venn Diagrams were obtained using raw data and filtering genes that were at least five-, four-, three-, and twofold upregulated when comparisons were made within each animal group between the two conditions, antigen challenge and saline challenge.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

Mast cell numbers and histological severity of antigen-induced cystitis.
We described previously that instillation of DNP4-OVA into the bladder of sensitized mice induced an inflammatory response characterized by cellular infiltration, plasma extravasation, and mast cell degranulation (24).

We now extend these observations by examining whether the bladders of mast cell deficient and control sensitized mice develop inflammation secondary to antigen challenge. Bladders isolated from sensitized +/+ mice challenged with saline did not present any sign of inflammation or edema (Table 1), while the bladders isolated from sensitized +/+ mice challenged with antigen exhibited an inflammatory response. At 24 h after antigen stimulation, the inflammatory response had predominantly characteristics of acute inflammation based on the strong vascular component, predominance of polymorphonuclear leukocytes (PMNs), and near absence of macrophages/monocytes. By contrast, histological examination of the bladder sections from KitW/KitW-v mice demonstrated a significant attenuation of the development of mucosal congestion and edema, as well as a reduction in the infiltration of PMNs in response to antigen challenge. Histologically, antigen-challenged bladders from sensitized KitW/KitW-v mice were similar to bladders isolated from sensitized +/+ mice exposed to saline. Bone marrow reconstitution of KitW/KitW-v mice repaired the mast cell deficiency in the urinary bladder (Fig. 1). Upon antigen challenge of sensitized BMR KitW/KitW-v mice,the inflammatory response was restored and statistically indistinguishable compared with antigen-challenged +/+ mice (Table 1).


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Table 1. Histological severity of antigen-induced cystitis

 


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Fig. 1. Histological evaluation of mast-cell reconstitution of KitW/KitW-v mice. Bone marrow cells derived from normal mice (+/+) were transferred intravenously into mast cell-deficient (KitW/KitW-v) mice. The animals were used for experiments 10 wk after reconstitution to allow mast cells to mature and acquire phenotypic characteristics normally seen in the urinary bladder. Giemsa-stained sections of the bladder were evaluated for mast cell number. A: bladder mucosa of +/+ mice. B: bladder detrusor muscle of +/+ mice. C: bladder mucosa of BMR. D: detrusor muscle of BMR mice. White arrows indicate mast cells, and black arrows indicate bladder urothelium. Original magnification, x40. The number of mast cells per cross section is presented in Table 1. BMR, bone marrow stem cell-reconstituted KitW/KitW-v mice.

 
Gene expression following acute inflammation.
These morphological alterations seen during antigen-induced cystitis appear to be dependent on the presence of mast cells, since they were not present in KitW/KitW-v mice but occurred when mast cells were reconstituted by bone marrow transplantation. However, the mechanistic information about the role of mast cells on tissue gene regulation during inflammation is limited. Therefore, we investigated whether the inflammatory response observed at the morphological level was accompanied by alterations in gene expression. For this purpose, three groups of mice were sensitized and challenged either with saline or antigen and killed 24 h after, as this is the time that coincides with the peak of the inflammatory changes (23). The expression of all 588 genes was normalized to ß-actin, and comparisons were made in tissues isolated from antigen- and saline-challenged mice.

To test the hypothesis that a particular group of genes exhibit differential expression during this inflammatory reaction, the cDNA array results obtained with all groups were calculated as a ratio of gene expression between antigen- and saline-treated groups.

Cluster analysis was performed using 588 genes, 6 groups (+/+, Wv, BMR treated with saline or antigen), and 2 hybridizations. A total of 7,056 data points were submitted to cluster analysis. We selected only four clusters based on strict criteria. Genes should be upregulated in +/+ and BMR and not in Wv. Filtering genes using GeneSpring software indicated that antigen challenge of sensitized +/+ mice resulted in an average of 2.1- to 5-fold increase in the expression of 29% of the genes present in the array. By contrast, antigen challenge of KitW/KitW-v mice resulted in increased expression in only 3.9% of genes. Antigen challenge of BMR of KitW/KitW-v mice led to an upregulation of 30% of the genes. Figure 2 shows a pictorial representation of the genes that were 4- to 2.0-fold upregulated by antigen challenge in each experimental group. The intersection between +/+ and BMR mice indicates genes that presented the same level of upregulation. The level of gene expression in response to antigen challenge and saline challenge in the different groups is also represented in a scatter plot (Fig. 3).



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Fig. 2. Venn diagram of ratio of gene expression (antigen/saline) obtained in bladder isolated from sensitized +/+, KitW/KitW-v, and BMR mice as determined by GeneSpring. Venn diagrams were obtained using raw data and filtering genes that were at least four-, three-, and twofold upregulated when comparisons were made within each animal group between the two conditions, antigen challenge and saline challenge. Results obtained with mast cell-deficient KitW/KitW-v mice (W/w-v) are in blue, congenic control mice (+/+) results are in red, and BMR KitW/KitW-v mice results are in green.

 


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Fig. 3. AC: expression profiling of all genes present in the array. For a typical hybridization, sensitized mast cell-deficient KitW/KitW-v (Wv, C), congenic control (+/+, A), and BMR KitW/KitW-v mice (B) were mice were used. Twenty-four hours after instillation with saline or antigen, bladders were dissected out and used for RNA extraction. Radiolabeled cDNAs from each sample were hybridized separately on the microarray. Two hybridizations were performed, one for each pooled bladder samples (n = 3), for a total of 6 mice. Expression of each gene was calculated as raw data, subtracted the background, and normalized as a percentage of ß-actin. The results of two hybridizations were averaged and displayed as scatter plot. The x- and y-axes represent gene expression as percentage of ß-actin in a log10 scale. Green lines indicate identical expression between the two treatments, and blue lines indicate twofold expression differences between antigen and saline.

 
Gene expression association with bone marrow reconstitution.
Out of 24 clusters, 4 fulfilled the criteria used for mast cell dependency of gene expression, as follows: 1) the genes were at least 2.0-fold upregulated in bladder isolated from antigen challenge of sensitized +/+ mice; 2) they were not altered in tissues isolated from antigen-challenged KitW/KitW-v mice, and 3) they were upregulated in bladders isolated from antigen-challenged BMR KitW/KitW-v mice. Cluster 1 contained a single gene, GABA-A transporter 3, that was fivefold upregulated by antigen challenge of +/+, not upregulated in KitW/KitW-v, and fivefold upregulated in BMR KitW/KitW-v mice (Fig. 4A; Table 2). Cluster 2 contained four genes that were upregulated in average of fivefold in +/+, not upregulated in KitW/KitW-v, and were recovered at least fourfold by BMR KitW/KitW-v mice (Fig. 4B; Table 2). Cluster 3 contained 13 genes that were upregulated in average of fourfold in +/+, not upregulated in KitW/KitW-v, and were recovered at least threefold by BMR KitW/KitW-v mice (Fig. 4C; Table 2). Cluster 4 contained 34 genes that were upregulated in average of threefold in +/+, not upregulated in KitW/KitW-v, and were recovered at least 2.1-fold by BMR KitW/KitW-v mice (Fig. 4D; Table 3).



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Fig. 4. AD: clusters 1–4. Mast cell-deficient KitW/KitW-v (Wv), congenic normal (+/+), and BMR KitW/KitW-v mice (BMR) were sensitized and challenged intravesically with either antigen or saline (see METHODS). Twenty-four hours later, the urinary bladders of 3 mice were isolated, homogenized, and processed for isolation of RNA. The RNA was labeled with [{alpha}-32P]dATP, reverse-transcribed to cDNA, and hybridized with membranes containing Atlas Mouse cDNA Expression Arrays. Radioactivity was quantified using a phosphor-imager and interpreted by OptiQuant image analysis software. Results of the expression of the 588 genes contained in the array were normalized to ß-actin expression. The array was repeated twice, and gene expression values were analyzed by GeneCluster software (http://www.genome.wi.mit.edu) using self-organizing maps (SOMs) as described before (53, 57). Twenty-four clusters were generated, and within each cluster results are presented as means ± SE of gene expression. The average values and SE represent the expression of GABA-A transporter 3 in two replicate experiments. A: cluster 1 contained a single gene, GABA-A transporter 3, that was fivefold upregulated by antigen challenge of +/+, not upregulated in KitW/KitW-v, and fivefold upregulated in BMR KitW/KitW-v mice. B: cluster 2 contained four genes that were upregulated in average of fivefold in +/+, not upregulated in KitW/KitW-v, and were recovered at least fourfold by BMR KitW/KitW-v mice. C: cluster 3 contained 13 genes that were upregulated in average of fourfold in +/+, not upregulated in KitW/KitW-v, and were recovered at least threefold by BMR KitW/KitW-v mice. D: cluster 4 contained 34 genes that were upregulated in average of threefold in +/+, not upregulated in KitW/KitW-v, and were recovered at least 2.1-fold by BMR KitW/KitW-v mice. *Statistically significant peak expression (P < 0.05). Numbers above each bar indicate the ratio of gene expression between antigen challenge and saline (SAL) challenge. Gene composition of each cluster is presented in Tables 2 and 3. The significance notation was performed using t-test for the comparison of the average of gene expression between saline- and antigen-treated tissues in two hybridizations as selected by cluster analysis. A: average of a single gene in two replications. B: average of four genes in two replications. C: average of 13 genes in two replications. D: average of 34 genes in two replications.

 

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Table 2. Clusters 1 and 2

 

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Table 3. Clusters 3 and 4

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Several lines of evidence suggest that mast cells play a role in bladder inflammation. The problem is to characterize and quantify the extent of the mast cells’ contribution to the response and to define the mechanisms by which mast cells influence these inflammatory reactions (8). By examining bladder inflammation in normal (+/+), mast cell-deficient KitW/KitW-v mice, and mast cell-deficient KitW/KitW-v mice that have had their mast cell deficiency corrected by bone marrow transplantation, we now provide evidence that mast cells contribute to antigen-induced bladder inflammation and gene expression. Normal (+/+) mice exhibited significant edema and neutrophil infiltration in the course of this inflammatory reaction, whereas mast cell-deficient KitW/KitW-v mice had little or no morphological evidence of inflammation. BMR of KitW/KitW-v mice completely restored the inflammatory response induced by antigen challenge. This strongly suggests that mast cells, or another bone marrow derived element(s), significantly participates in the development of bladder inflammation in this response. The mast cell is the most likely immune component to explain this response, since immunologic responses that do not require mast cells are intact in KitW/KitW-v mice (30). However, the precise role of the mast cell can only be answered in a definitive manner by reconstituting KitW/KitW-v mice selectively with mast cells. This approach has been used to examine mast cell responses in the skin, stomach, and small intestine of mice (2931) but has not been studied in the urinary bladder. Further studies will be necessary to extend this model of selective mast cell repletion to the bladder of the mouse.

The participation of mast cells in mucosal inflammatory responses has been controversial. Wershil et al. (29) reported that mast cells were required for the infiltration of neutrophils and mononuclear cells into the gastric wall during IgE-dependent inflammation in a model of passive sensitization. By contrast, in an actively sensitized, antigen-induced model of intestinal inflammation, they found that mast cells were not required for the development the response in the intestinal tract (5). Our findings in the urinary bladder suggest an essential role for mast cells in a model of actively sensitized bladder inflammation. The reason for the differences in mast cell dependency between the intestinal tract and the urinary bladder is not clear. It may be related to the sensitization protocols, method of delivery of antigen, or some fundamental difference in IgE-dependent responses at these mucosal surfaces.

In addition to the inflammatory response, we also examined tissue gene expression using a gene array strategy. We previously presented evidence of the reproducibility of gene array methodology for the analysis of bladder inflammatory genes (23) and verified the results using RNase protection assay (23). Using this approach, we identified a subset of genes that were expressed upon antigen stimulation in normal mice but not mast cell-deficient KitW/KitW-v mice. Moreover, the elicitation of the inflammatory response in BMR KitW/KitW-v mice resulted in the expression of these genes as occurred in normal mice. Thus we were able to identify a variety of genes that required mast cells for their expression during antigen-induced bladder inflammation. For example, IL-6, macrophage inflammatory protein-2{alpha} (MIP-2{alpha}), IL-4 receptor (IL-4R, membrane-bound form), integrin-{alpha}2 (CD49b), integrin-{alpha}4, integrin-{alpha}5 (CD51), intercellular adhesion molecule-1 (ICAM-1), mitogen-activated protein kinase kinase (MAPKK), and thrombopoietin (TPO) were all upregulated in a mast cell-dependent manner. The mast cell dependency of the expression of a given gene implies that either the mast cell is expressing the gene and/or the mast cell is inducing the expression of the gene by another cell type. Further studies will be necessary to define the cell types expressing these genes.

It should be noted that we only analyzed a single time point due to the complexity of this study, i.e., employing three types of mice and two different treatments. Our previous studies indicated that although some genes, such as proto-oncogenes, have a transient early expression, the vast majority of genes that are upregulated maintained an enhanced level of expression up to 24 h after the stimulation (23). It will be important to examine a time course of antigen-induced gene regulation in the future.

Also, to fairly interpret gene cluster analysis, it is important to account for a growing body of evidence that gene and protein changes can be dissociated (6, 10, 28). For instance, increased abundance of urinary bladder nerve growth factor mRNA is not always associated with increased total urinary bladder nerve growth factor (28). The discrepancy between two measures (mRNA and protein) may reflect retrograde axonal transport of nerve growth factor to the dorsal root ganglia (28). Future proteomic correlation must determine how directly mRNA changes reflect translated protein levels and the physiological consequence of these proteins (3). In particular, gene cluster analysis may allow us to begin to understand clinically relevant issues, especially how and why the transition from acute to chronic inflammation occurs only in selected circumstances and which pathophysiological and therapeutic strategies change the normal balance of apoptosis and necrosis in populations of bladder cells.

In conclusion, our findings demonstrate an important role for mast cells in antigen-induced bladder inflammation and gene expression. These genes are strong candidates for mediating this mast cell-dependent inflammatory response. It remains to be determined whether mast cells themselves are expressing these mRNAs or whether the mast cells are inducing their expression by another cell type. Finally, this work defines a new approach for analyzing how mast cells express their function, as well as for investigating the roles of specific gene products (or agents that enhance or suppress their activity) in host defense, tissue remodeling, and models of cystitis.


    ACKNOWLEDGMENTS
 
This research was supported by National Institutes of Health Grants DK-55828 (to R. Saban), DK-51392 (to T. G. Hammond), and DK-46819 and DK-33506 (to B. K. Wershil).


    FOOTNOTES
 
Article published online before print. See web site for date of publication (http://physiolgenomics.physiology.org).

Address for reprint requests and other correspondence: R. Saban, Associate Professor, Dept. of Physiology, College of Medicine, Oklahoma Univ. Health Science Center, 940 SL Young Blvd. Rm. 605, Oklahoma City, OK 73104 (E-mail: ricardo-saban{at}ouhsc.edu; http://moon.ouhsc.edu/rsaban/).


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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