A mutant collagen XIII alters intestinal expression of immune response genes and predisposes transgenic mice to develop B-cell lymphomas

Abstract

Epithelial cells of mucosal surfaces are critical for maintaining immune homeostasis by aiding in the discrimination of pathogenic and commensal microorganisms and modulating the activities of antigen-presenting cells and lymphocytes. Functional breakdowns resulting in chronic infection and inflammation are associated with the development of hematologic and solid neoplasms for which detailed pathogenetic mechanisms are poorly understood. Mice heterozygous for a transgene Col13a1(del) expressing a mutant collagen XIII developed clonal mature B-cell lineage lymphomas originating in mesenteric lymph nodes (MLN). The tumors were associated with T cells and macrophages. The incidence of disease was reduced 2-fold in transgenic mice raised under specific pathogen-free conditions, suggesting a role for infectious agents. The lymphomas did not express the mutant collagen XIII, indicating that its influence on tumorigenesis was B-cell extrinsic and likely to be associated with collagen XIII-positive tissues drained by the MLN. Studies of the small intestines of transgenic mice showed that the subepithelial basement membranes (BM) were highly abnormal and that they exhibited heightened expression of genes involved in immune responses. These results define collagen XIII-dependent maintenance of the intestinal BM as a previously unappreciated component of immune responses and a critical determinant of cancer susceptibility.

Figure 1

Analysis of the lymphomas of the intestine. A) Macroscopic appearance of a mesenteric lymphoma. B–C) According to the FACS analysis varying proportions of the cells were found to express the T cell antigen, CD3, (B) or the B cell marker CD19 (C). The histograms are gated on total viable lymphocytes and the numbers in B and C are the percentages of CD3 positive T cells and CD19 positive B cells of these cells. D) Southern blot analysis of the lymphomas revealed clonal immunoglobulin gene rearrangement (lanes 1–3, three individual lymphoma cases). The HpaI digested 16.4 kb germ line arrangement is indicated by an arrow and the rearranged forms by arrowheads.

Figure 2

Histological analysis of tumor formation. A) Histology of pre-neoplastic spleen and MLN. Spleen (A1, x200) from 12-month-old transgenic mouse showing a central arteriole (indicated by asterisk) and surrounding white pulp and adjacent active germinal center (diamond) with preneoplastic changes. In addition to normal small lymphocytes, the periarteriolar lymphoid sheath (PALS) is filled with larger lymphocytes (arrows). In an active germinal center, these large lymphocytes extend into the red pulp area resulting in a GC with irregular borders (arrowheads). The higher magnification (A2, x400) shows infiltrating centroblasts (dark arrowheads), lymphoblast-like cells (arrows) and histiocytes (open arrowheads) in the PALS area where the central arteriole is indicated by an asterisk. In a more advanced case, both the spleen (not shown) and lymph nodes (A3, x200) exhibit enlarged follicles that appear to expand from the GC-like structures in the nodes, trapping the few residual normal small lymphocytes in the cords (arrows) and finally resulting in the nearly complete obliteration of normal lymph node architecture shown in figure A4 with smaller magnification (x40). B) Histology of tumors of the intestine. Immunoblastic lymphoma (B1, x1000) with plasmacytoid differentiation consisting of immunoblasts (arrowheads), plasmablasts (arrows) and plasma cells scattered among a sea of histiocytes with a pink granular cytoplasm (open arrowheads). The dominance of the histiocyte population is readily seen at lower power (B2, x400). Immunoblastic lymphoma (B3, x400) with accumulation of histiocytes arrayed in a pseudo-rosette pattern. Figure B4 shows histiocytic accumulation (arrowheads) without an associated lymphoma (x400). C) The tumors were positive for B cell marker B220 (C1), but negative for collagen XIII (Col XIII) (C2). Scale bar 50 µm in panel C, as indicated in C1.

Figure 3

Expression of the Col13a1^del transgene and endogenous collagen XIII in the lymphomas and intestine. A) RT-PCR analysis of the expression of the transgene (tg, 297 bp) and the endogenous collagen XIII gene (Col XIII, 567 bp) in several lymphomas (lanes 1–5) from two transgenic lines, in normal MLN in control (lane 6) and transgenic mouse (lane 7) and in the large intestine of the transgenic mice (lane 8) studied by Southern blotting. Lane 9 is a negative control for RT reaction and lane 10 negative control for PCR reaction. The selected primers allowed simultaneous amplification of both the endogenous and transgenic mRNAs. Transgene expression is equal to or higher than endogenous collagen XIII expression in all tumors studied. B) RT-PCR analysis showed that endogenous collagen XIII mRNA was highly expressed both in large (lane 3) and small intestine (lane 4) of control mice. Both the transgene and endogenous collagen XIII were clearly expressed in large (lane 1) and small (lane 2) intestine of Col13a1^del mice at comparable level. Lane 5 is a negative control for RT reaction and lane 6 negative control for PCR reaction. GAPDH was used as an internal control.

Figure 4

Collagen XIII expression in the Peyer’s patches, small intestine and spleen. A) Collagen XIII is not expressed by the lymphocytes (arrowheads) within the Peyer’s patches in either the wild type (A3) or Col13a1^del (A4) mouse intestine as identified with the B cell marker CD45R (A1,A2) and type IV collagen staining (A5,A6). Only non-specific staining (marked with an asterisk) is detected. B) Strong collagen XIII staining is localized underneath the epithelial cell layer in the villus area of the small intestine both in control (B1) and in Col13a1^del mice (B2), and also adjacent to the Peyer’s patch (A3,A4). In addition to the epithelial BM, collagen IV is localized in lamina propria around the blood vessels (B3, B4). C) Both in wild type and Col13a1^del mice collagen XIII (C1,C2) is co-localized with PECAM (C3,C4) in the small vessels of the spleen, whereas the large vessels (marked with an asterisk) are negative for collagen XIII. D) Collagen XIII (D1), PECAM (D2) and desmin (D3) staining in serial sections of the wild-type spleen suggests a pericyte localization of collagen XIII. Scale bar 100 µm in panels A and B, as indicated in A1, 50 µm in panel C, as indicated in C1, and 20 µm in panel D, as indicated in D1.

Figure 5

Transmission electron microscopy of the small intestine. Where the epithelial cells of the small intestines of control mice (A) showed well-defined BM (asterisk), the Col13a1^del mice (B) had abnormal assembly of the BM (asterisk) with an absence of the lamina lucida. Abbreviations used: EC, epithelial cell; EDC, endothelial cell; FB, fibroblast. Bars, 200 nm.