Final Progress Report

Proposal No.   IBD-0066
Principal Investigator:  Judith Bond, Ph.D. (replacement PI);   Jacqueline Crisman, Ph.D. (original PI)
Applicant Organization:  The Pennsylvania State University, College of Medicine (Hershey, U.S.A.)
Project Title: Intestinal leukocyte infiltration is mediated by meprin beta
Period of Award:  June 1, 2003 – July 31, 2004

A.  Summary of project aims.

The two aims of this grant were:

(1) To assess the role of the meprin ß metalloprotease in the dissemination of macrophages of the normal intestinal immune system, and

(2) To assess the role of meprin ß in the dissemination of macrophages of the intestinal immune system after induction of sodium dextran sulfate (DSS)-mediated intestinal inflammation

B.  Accomplishments towards meeting those aims.

The results of these investigators support our hypothesis that the metalloprotease meprin ß plays an active role in intestinal pathophysiology by facilitating macrophage infiltration into the intestinal immune system.  Meprins have been primarily studied in the kidney and intestine as brush border membrane enzymes, however recently there was a report indicating that meprins were in the lamina propria of the human inflamed bowl.  Our studies established that meprins are expressed in leukocytes of the mouse mesenteric lymph node, and we were the first to investigate meprin proteases in an inflammatory mouse model.

The results of our studies indicated:

(1) meprin a and ß mRNA are present in mesenteric lymph nodes of untreated and DSS-treated mice,

(2) meprin proteins are detected by immunofluorescence in untreated and DSS-treated mice,

(3) meprin a protein, but not ß, was decreased in mesenteric lymph nodes from DSS-treated mice compared with that from untreated mice,

(4) meprins are expressed in macrophages of the mesenteric lymph node, and

(5) leukocytes from meprin ß null mice are deficient in their ability to invade into matrigel.

These studies have been published (Crisman JM, Zhang B, Norman LP, Bond JS (2004) Deletion of the mouse meprin ß metalloproteinase gene diminishes the ability of leukocytes to disseminate through extracellular matrix.  J Immunol 172:4510-4519).

During the last six months, a system has been developed to further test the hypothesis that meprin plays a role in intestinal inflammation in vivo.  Intestinal inflammation was induced by oral administration of DSS in mice, generating an experimental model for inflammatory bowel disease (IBD).  Mice (9 – 10 weeks old) were given 3.5% DSS in the drinking water for four to seven days.  The results showed that wild-type mice (C57Bl/6x129) had a more severe reaction to DSS than meprin ß null mice on the same genetic background as determined by body weight loss, intestinal bleeding, mortality and lethargy.  Histological examination of the colon and ileum is currently ongoing.  The results of these studies showing a less severe response to DSS in the ß knockout mice are consistent with our hypothesis that meprin ß plays a role in the pathogenesis of IBD.

In addition, we have observed differences in the intestinal flora of the wild-type and meprin ß knockout mice.  In the presence of DSS, a shift in the facultative intestinal flora of the wild-type mice from Gram positive to Gram negative occurs; this is not seen in the meprin ß knockout mice.  Because the bacterial flora are implicated in the development of intestinal inflammatory diseases, these are important observations that are being pursued.

To specifically monitor migration of leukocytes in vivo, the original plan was to fluorescently label leukocytes from meprin ß null or wild type mice, remove the labeled leukocytes from lymph nodes, and administer them intravenously to wild type and meprin ß null recipients.  It was proposed to examine the deposition of fluorescent leukocytes in intestinal tissues using flow cytometry and immunofluorescence.  Unfortunately, the fluorescence of the administered leukocytes (using a fluorescein derivative) was too low to measure effectively.  Therefore, a new strategy was devised.  Transgenic mice carrying an enhanced green fluorescent protein (EGFP) expressed by a class I promoter were produced.  These mice were then mated with meprin ß null or control mice.  The EGFP (+) leukocytes from wild-type mice have now been used to perform passive transfer studies in wild-type mice.  The initial experiments show that leukocytes can be followed after administration into host mice.  Preliminary data are shown in the figure at the end of this report.  The EGFP(+) mice have been crossed with meprin ß heterozygotic mice and the pups genotyped.  It has taken longer than anticipated to generate a sufficient number of mice to conduct the passive transfer experiments.  However, we are now ready to administer the EGRP-labeled leukocytes from wild-type and meprin ß null mice to hosts.  The hosts for these studies will include control mice and mice treated with DSS.  The studies will establish whether the meprin ß metalloprotease actively participates in the deposition of leucocytes into the intestine under normal and inflammatory conditions.

C.  List of significant results (positive or negative).

(1) meprin subunits (α and ß) are expressed in mouse mesenteric leukocytes; the subunits are in different cell types

(2) meprin a, but not ß, expression decreases in mesenteric lymph nodes during inflammation

(3) meprin ß is present in macrophages of lymph nodes

(4) deletion of the meprin ß gene decreases the migration of leukocytes through matrigel

(5) meprin ß null mice are less vulnerable than wild-type mice to intestinal damage by DSS

(6) meprin ß null mice have a different profile of bacterial flora in the intestine than wild-type mice

(7) mice expressing a green fluorescent protein have been crossed with wild-type and meprin ß null mice to do passive transfer studies to determine whether meprin ß enhances the ability of macrophages to migrate to the intestine during inflammation.

D.  Lay summary of this report

Our immune system is critically important for destroying foreign material in our bodies.  However, sometimes there is an overreaction of this system that leads to destruction of our own tissues, leading to diseases such as inflammatory bowel disease (IBD).  We are using an experimental mouse model to study IBD and factors that lead to uncontrolled destruction of intestinal tissue with the aim of controlling the destruction.  When an intestinal irritant such dextran sulfate sodium (DSS) is administered to mice, there is an immune response where white blood cells move into the bowel and cause severe tissue damage.  Our studies indicate that a protease call meprin is one of the genes responsible in the destructive cells that allow too many white blood cells to migrate into the intestine.  When the gene for meprin is eliminated in mice, there is a much less severe response in the intestine to DSS.  White blood cells that have the meprin gene deleted do not move through a matrix of proteins that surround our cells, and we propose that without the meprin protein the migration of the destructive cells is compromised.  This work provides insights into a mechanism that tissue-destroying cells use to move toward a site of inflammation.  This information can lead to therapies to interfere with meprin activities and lessen the damage to our tissues in inflammatory conditions.

 Graphic

This figure demonstrates that enhanced green fluorescent protein EGFP(+) spleenocytes from transgenic EGFP mice migrate to the mesenteric lymph node of host mice and can be readily detected.  The mesenteric lymph nodes of the host show the green fluorescent protein (green), and an antibody to GFP (red) are colocalized.  The blue color represents nuclei stained with Hoechst 33342.  Magnification: 60x.