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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online September 9, 2004 as doi:10.1096/fj.04-1855fje. |
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Centre for Cellular and Molecular Biology, Hyderabad, India
1Correspondence: E-mail: khar{at}ccmb.res.in
SPECIFIC AIMS
We have demonstrated a novel route of migration of immune cells into the peritoneum after subcutaneous (s.c.) transplantation of AK-5 tumor. We have shown migration of APCs from the subcutaneous site to the draining lymph node and into the peritoneum. Activated migratory APCs perform their antigen presentation function in the peritoneum leading to heavy influx of immune cells in the peritoneum. These cells are activated in the peritoneum and migrate to the tumor where they induce tumor cell death. MIP-3ß produced by peritoneal mesothelial cells is involved in attracting immune cells. Other chemokines such as MIP-1
, MIP-3, IP-10, and lymphotoxin- are involved in immune cell migration whereas IL-2, IL-12, and IFN-
induce activation of these cells. Our observations suggest that the migration phenomenon is universal and that the peritoneum functions as a lymphoid organ involved in enhancement of effector cell function.
PRINCIPAL FINDINGS
1. Antigen injection at the subcutaneous site causes cellular influx into the peritoneum
Transplantation of AK-5 tumor cells s.c. lead to heavy influx of immune cells into the peritoneum around day 15 (Fig. 1
). Immune cells were activated and migrated to the tumor where they were involved in causing tumor cell death. This phenomenon was found to be relevant to general immune response since other inflammatory stimuli such as Escherichia coli, leishmania parasites, Meth A tumor cells, and AK-5 tumor cell transplantation in nude mice also induced immune cell migration into the peritoneum (Fig. 1)
.
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2. Chemokine up-regulation induced migration of cells
Molecules involved in the attraction of immune cells into the peritoneum and back to the tumor site were investigated. AK-5 tumor expressed chemokines MIP-1 and MIP-3, whereas the peritoneal mesothelial cells expressed lymphotoxin- and MIP-3ß (Fig. 2
). These chemokines were up-regulated upon antigen injection. Tumor tissue secreted MIG/CXCL9 and IP-10/CXCL10, the induction of which was stimulated by IFN-. Cytokines IL-2, IL-12, and IFN- present in peritoneal fluid are involved in the activation of immune cells, whereas IFN- could influence cellular migration.
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3. Migration of cells to the lymph node and peritoneum
Migration of cells from the subcutaneous site to the neighboring lymph nodes and the peritoneum was monitored using cell tracking dye PKH-26. Tumor cells were labeled with the dye in vitro and injected s.c. PKH-26 fluorescence was monitored in neighboring lymph nodes and in the peritoneum on different days. Lymph nodes were positive by day 1, whereas fluorescent cells were seen in the peritoneum by day 3. Migration of tumor cells to these sites was ruled out. Similar observations were made for antigen presenting cells (i.e., when DCs and macrophages were labeled with cell-tracking dye). Leishmania parasites and other antigens induced similar migration of cells. On the other hand, no migration was observed in the absence of antigen.
4. Identity of migratory cells
To establish the nature of cells migrating from the subcutaneous site to the peritoneum, the PKH-26-labeled cells from the peritoneum were allowed to attach to glass coverslips. Attached cells were stained with OX-41 (M
marker) and OX-62 (DC marker). Results confirmed migratory cells to be either Ms or DCs. Other markers such as CD86 and MHC class II were present on migratory cells.
5. Function of migratory cells
The role of migratory cells in the peritoneum was established using lymphoproliferation assays. Two sets of animals were immunized with either ovalbumin or tumor rejection antigen isolated in the lab. Peritoneal cells on different days after the antigen injection were tested in lymphoproliferation assays using purified antigen. Significant lymphoproliferation was achieved in day 14 and day 15 peritoneal immune cells, confirming our prediction that the migratory cells are involved in antigen presentation function in the peritoneum.
CONCLUSIONS AND SIGNIFICANCE
Host defense and immune surveillance are highly dependent on mobile leukocytes that form continuously renewed microenvironments and can be rapidly recruited to the site of inflammation. Priming and activation of immune cells against any antigenic stimuli is an important prelude to immune attack. After a pathogen or tumor invasion, APCs are recruited to the site of inflammation. Immature APCs migrate in response to MIP-3 and MIP-1, also expressed at the tumor site (Fig. 2A
). APCs attain maturity by losing their response to MIP-3 when they attain a sustained responsiveness for MIP-3ß. APCs are known to be selectively recruited into lymphoid organs by lymphotoxin- which is expressed by peritoneal mesothelial cells (Fig. 2B
).
Expression of MIP-3ß is restricted to lymphoid organs and to cells of hematopoietic origin. MIP-3ß expression is induced in peritoneal mesothelial cells and macrophages (Fig. 2D
). Once APCs have captured antigens and migrate to lymph nodes, they mature when their surface expression of CD86 and MHC class II are highly increased.
Mature APCs migrated to the peritoneum where they performed antigen presentation function as evaluated by lymphoproliferation assays. We found migratory APCs to be of dendritic and macrophage lineage. Accumulation of chemokines IL-12 and IFN- in the peritoneum induced influx of immune cells into the peritoneum, where they are activated. We have shown migration of activated cells from the peritoneum to the tumor site where they participate in inducing death in tumor cells. Migration of APCs to the peritoneum seems to be a universal phenomena since other antigens such as bacteria, parasites, and other tumors induced similar migration. No migration of either APCs or immune cells into the peritoneum took place when animals were injected with syngeneic hepatocytes at the subcutaneous site. These observations suggest that the peritoneum acts as an important lymphoid organ where presentation of antigen to the immune cells takes place and is followed by their migration to the inflammatory site (Fig. 3
).
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FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-1855fje;
This article has been cited by other articles:
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  R. M. Srivastava, Ch. Varalakshmi, and A. Khar Cross-linking a mAb to NKR-P2/NKG2D on dendritic cells induces their activation and maturation leading to enhanced anti-tumor immune response Int. Immunol., May 1, 2007; 19(5): 591 - 607. [Abstract] [Full Text] [PDF]
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