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. 2001 Oct;49(4):526–533. doi: 10.1136/gut.49.4.526

MIP-2 secreted by epithelial cells increases neutrophil and lymphocyte recruitment in the mouse intestine

Y Ohtsuka 1, J Lee 1, D Stamm 1, I Sanderson 1
PMCID: PMC1728474  PMID: 11559650

Abstract

BACKGROUND—Invasion of the intestinal mucosa by leucocytes is a characteristic of intestinal inflammation but the role of the epithelium in orchestrating this recruitment has not been examined in vivo. Cultured intestinal epithelial cells secrete a wide variety of chemokines, often in response to agents present in the intestinal lumen. Macrophage inflammatory protein 2 (MIP-2) is a chemokine that attracts neutrophils, and its secretion from intestinal epithelial cells is enhanced by inflammatory stimuli such as interleukin 1β. We hypothesised that the production of MIP-2 by epithelial cells would increase leucocyte migration into the intestine.
AIM—To study the effects of a chemokine secreted from intestinal epithelial cells in vivo.
METHODS—MIP-2 was expressed in the mouse intestinal epithelium using an epithelial cell specific promoter from the gene encoding the intestinal fatty acid binding protein. The intestines of these transgenic mice were then analysed.
RESULTS—Epithelial cells from transgenic mice expressed MIP-2 but wild-type mice did not. Neutrophil recruitment, examined by myeloperoxidase (MPO) staining and total MPO activity per unit weight of intestine, was significantly increased in transgenic mice in both the small intestine and proximal colon, and this was blocked by anti-MIP-2 antibody treatment. Both intraepithelial and lamina propria lymphocytes were also increased in transgenic mice. They showed chemotactic activity to MIP-2 in the Boyden chambers and expressed MIP-2 receptor (CXCR-2) mRNA confirmed by reverse transcription-polymerase chain reaction.
CONCLUSION—These experiments are the first to show a functional role for epithelial chemokines in vivo and reveal an unexpected role for the neutrophil chemokine MIP-2 in controlling mucosal lymphocyte migration.


Keywords: chemokines; epithelial cells; intestinal mucosa; leucocyte recruitment; transgenic mouse

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Figure 1  .

Figure 1  

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Generation of macrophage inflammatory protein 2 (MIP-2) transgenic mice. (A) Diagrammatic representation of the plasmid containing the fatty acid binding protein of the intestine (Fabpi) promoter linked to the MIP-2 gene and the SV40 intron and polyadenylation site. MIP-2 cDNA was derived from a mouse MIP-2 sequence using polymerase chain reaction with primers synthesised with a Xho I site and an Xba I site. (B) Northern blot analysis of MIP-2 mRNA accumulation in the intestinal epithelium of wild-type and transgenic mice. MIP-2 mRNA was detected using an MIP-2 cDNA probe. Epithelial cells were removed from wild-type mice (lanes 1, 2) and from transgenic mice (lanes 5, 6). MIP-2 mRNA derived from IEC-6 cells stimulated with interleukin 1β (IL-1β) was used as a positive control (lanes 3, 4).

Figure 2  .

Figure 2  

Figure 2  

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Neutrophil recruitment increases in macrophage inflammatory protein 2 (MIP-2) transgenic mice. (A) Neutrophils in the mid jejunum were stained by myeloperoxidase (MPO) using the Hanker Yates' reaction. Magnification ×100. (B) Neutrophil recruitment is demonstrated by total MPO activity. MPO was extracted with 0.5% hexadecyltrimethylammonium bromide. Total MPO activity is expressed per unit weight of tissue derived from wild-type MIP-2 transgenic mice. Expression of MPO activity as a proportion of unit per weight of whole tissue appeared to be smaller in the small intestine than in the colon due to the heavier weight per unit length of the small intestine. Data are presented as mean (SD) of eight animals for each group. **p<0.01 versus wild-type.          

Figure 3  .

Figure 3  

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Neutrophil recruitment due to macrophage inflammatory protein 2 (MIP-2) secretion was blocked with an anti-MIP-2 antibody. Anti-MIP-2 antibody was injected intravenously seven days and four days before examining the mice. Neutrophil recruitment was examined by measuring myeloperoxidase (MPO) activity per unit weight of tissue samples derived from wild-type and MIP-2 transgenic mice. Ab (−), mice received phosphate buffered saline only; Ab (+), mice received anti-MIP-2 antibody treatment. Data are presented as mean (SD) of eight animals for each group. **p<0.01.

Figure 4  .

Figure 4  

Figure 4  

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Intraepithelial lymphocyte (IEL) and lamina propria lymphocyte (LPL) numbers were increased in the small intestine of macrophage inflammatory protein 2 (MIP-2) transgenic mice. (A) Lymphocytes were detected with an anti-CD3 Ab (145-2C11). Magnification ×100. (B) IELs were expressed as number of CD3 positive cells in the epithelium per 100 epithelial cells. LPLs were expressed as CD3 positive cells in the lamina propria per mm2. Data are presented as mean (SD) of six animals for each group. *p<0.05, **p<0.01.

Figure 5  .

Figure 5  

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Macrophage inflammatory protein 2 (MIP-2) attracts lymphocytes, including intraepithelial lymphocytes (IEL) in vitro. Lymphocyte migration in response to interleukin 10 (IL-10) and MIP-2 in the absence or presence of anti-MIP-2 antibody (MIP-2/Ab) was studied using the Boyden chamber. Lymphocytes (2×105/0.2 ml) were placed in the upper chambers, and migrated cells were counted after four hours of incubation at 37°C. Each experiment was done in duplicate. Data are presented as mean (SEM) of six animals for each group. Significant differences were evaluated in assays between supernatants with or without anti-MIP-2 antibody. **p<0.01.

Figure 6  .

Figure 6  

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Analysis of CXCR-2 expression on lymphocytes from wild-type mice. Agarose gel stained with ethidium bromide showing reverse transcription-polymerase chain reaction (RT-PCR) products for CXCR-2 (331 bp) and GAPDH (423 bp). Expression of CXCR-2 and GAPDH mRNA was confirmed in intraepithelial lymphocytes (IEL) and lamina propria lymphocytes (LPL) isolated from wild-type mice. RNA extracted from whole spleen was used as a positive control and PCR mixture without RNA but distilled H2O was used as a negative control. Representative of three independent experiments.

Selected References

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