scientificprotocols authored about 6 years ago
Authors: Jennifer Gommerman & Jorg Fritz
The largest mucosal surface in the body is in the gastrointestinal (GI) tract, a location that is heavily colonized by normally harmless microbes. A key mechanism required for maintaining a homeostatic balance between this microbial burden and the lymphocytes that densely populate the GI tract is the production and trans-epithelial transport of poly-reactive IgA1. Within the mucosal tissues, B cells respond to cytokines, sometimes in the absence of T cell help, undergo class switch recombination (CSR) of their Immunoglobulin (Ig) receptors to IgA, and differentiate to become plasma cells (PC)2. However, IgA-secreting PC likely have additional attributes that are needed for coping with the tremendous bacterial load in the GI tract. We report that IgA+ PC also can produce the anti-microbial mediators TNFα and iNOS, which appear to arise in the unique environment of the gut, and may be critical to mount effective responses to microbial assault.
Here we described a detailed method to characterize and quantify IgA+iNOS+TNFα+ cells that we called TNFα-iNOS-producing (Tip)-PC in the lamina propria of mice by immunohistochemistry (IHC).
The largest mucosal surface in the body is in the gastrointestinal (GI) tract, a location that is heavily colonized by normally harmless microbes. A key mechanism required for maintaining a homeostatic balance between this microbial burden and the lymphocytes that densely populate the GI tract is the production and trans-epithelial transport of poly-reactive IgA1. Within the mucosal tissues, B cells respond to cytokines, sometimes in the absence of T cell help, undergo class switch recombination (CSR) of their Immunoglobulin (Ig) receptors to IgA, and differentiate to become plasma cells (PC)2. However, IgA-secreting PC likely have additional attributes that are needed for coping with the tremendous bacterial load in the GI tract. We report that IgA+ PC also can produce the anti-microbial mediators TNFα and iNOS, which appear to arise in the unique environment of the gut, and may be critical to mount effective responses to microbial assault.
Here we described a detailed method to characterize and quantify IgA+iNOS+TNFα+ cells that we called TNFα-iNOS-producing (Tip)-PC in the lamina propria of mice by immunohistochemistry (IHC).
Antibodies:
Harvest tissue and preparation of sections will require approximately four hours. The staining of tissue sections will then take approximately five hours.
Applying this staining methods we observes that IgA expression co-localizes with the expression of iNOS and TNFα with some, but not all IgA+ cells in the small intestinal LP tissue. Occasional co-expression of both iNOS and TNFα was observed in IgA+ cells, consistent with our flow cytometry data. Taken together, we have found that a proportion of IgA+ cells within the small intestinal LP express anti-microbial mediators TNFα and iNOS, while we do not detect significant expression of these molecules in other LP cells.
1 Hooper, L. V. & Macpherson, A. J. Immune adaptations that maintain homeostasis with the intestinal microbiota. Nat Rev Immunol 10, 159-169, (2010). - Fagarasan, S., Kawamoto, S., Kanagawa, O. & Suzuki, K. Adaptive immune regulation in the gut: T cell-dependent and T cell-independent IgA synthesis. Annu Rev Immunol 28, 243-273, (2010).
We thank Dionne White in the Faculty of Medicine Flow Cytometry core facility. C.P. is supported by a CIHR operating grant MOP# 9862. R.C. is supported in part by the Intramural Research Program of the National Institute of Arthritis and Musculoskeletal and Skin Diseases of the National Institutes of Health. A.M. is supported by a CIHR operating grant MOP# 89783. J.H.F. acknowledges support by an APART-fellowship of the Austrian Academy of Sciences, McGill start-up funds and a CIHR operating grant MOP#114972. N.S. acknowledges the support of a CIHR Doctoral Award. J.L.G. is funded by the Canadian Institutes of Health Research (CIHR) and acknowledges the support of CIHR operating grant MOP# 67157 as well as infrastructure support from the Ontario Research Fund and that Canadian Foundation for Innovation.
Protocol with Figure 1.: Detection of TNF/iNOS/IgA in the small intestinal lamina propria by IF
Download Protocol with Figure 1.
Acquisition of a multifunctional IgA+ plasma cell phenotype in the gut. Jörg H. Fritz, Olga Lucia Rojas, Nathalie Simard, Douglas D. McCarthy, Siegfried Hapfelmeier, Stephen Rubino, Susan J. Robertson, Mani Larijani, Jean Gosselin, Ivaylo I. Ivanov, Alberto Martin, Rafael Casellas, Dana J. Philpott, Stephen E. Girardin, Kathy D. McCoy, Andrew J. Macpherson, Christopher J. Paige, and Jennifer L. Gommerman. Nature doi:10.1038/nature10698
Jennifer Gommerman, Gommerman Lab, University of Toronto
Jorg Fritz, McGill University
Correspondence to: Jorg Fritz ([email protected])
Source: Protocol Exchange (2011) doi:10.1038/protex.2011.270. Originally published online 16 December 2011.