Isolated lymphoid follicle maturation induces the development of follicular dendritic cells

Abstract

Isolated lymphoid follicles (ILFs) are recently identified lymphoid structures in the small intestine with features similar to Peyer's patches (PPs). Using immunohistochemistry we characterized the composition of ILFs in the small intestines of immunocompetent mice and of mice that lacked PPs as a result of either genetic deficiency of lymphotoxin or temporary in utero lymphotoxin-beta receptor-signalling blockade. We showed that although both immature and mature ILFs were present in the intestines of immunocompetent mice, PP-deficiency induced a significantly greater number of mature ILFs. We found that in addition to B-lymphocyte-containing germinal centres, mature ILFs also possessed large networks of follicular dendritic cells (FDCs). These features were not detected within immature ILFs. Indeed, the presence of FDCs could be used to reliably distinguish ILF maturity. Further analysis revealed that the area occupied by the FDCs within mature ILFs was substantial. The total area occupied by FDCs in all the mature ILFs in mice lacking PPs was equivalent to the total area occupied by FDCs in all the PPs and the few mature ILFs in immunocompetent mice. Based on these data we reasoned that in the absence of PPs, mature ILFs are important inductive sites for intestinal immune responses. Indeed, in mice that lacked PPs, ILF maturation coincided with a restoration of faecal immunoglobulin A levels to values that were comparable to those found in immunocompetent mice. Taken together, these data imply that the induction of germinal centres and FDC networks within mature ILFs in response to PP deficiency provides an important compensatory mechanism.

Figure 1

Detection of B lymphocytes and FDCs within ILFs and PPs in the small intestine. Entire small intestines were examined from each mouse group and representative images are shown. Sections were stained with the CD45R-specific monoclonal antiserum B220 to detect B lymphocytes (green) and 7G6 monoclonal antiserum to detect CR2/CR1 (CD21/35) expressing FDCs (red). In each mouse group analysed, FDCs were absent within the B-lymphocyte aggregations of immature isolated lymphoid follicles (iILFs; left column), whereas both B lymphocytes and FDCs were detected within all mature ILFs (mILFs; middle column). Original magnification ×200.

Figure 2

Immunohistochemical characterization of FDCs within mILFs. Sections of small intestine or spleen (far right column) were stained with CD45R-specific monoclonal antiserum B220 to detect B lymphocytes (top row, green), CR2/CR1 (CD21/35)-specific (top row, red) or CR1 (CD35)-specific monoclonal antisera to detect FDCs (second row, red) and monoclonal antisera specific for complement components C4 (third row, red) and C3 (bottom row, red). Complement components in association with CR1- and CR2-expressing FDCs were detected in both mILFs and the spleen. Original magnification ×200.

Figure 3

Enumeration of ILFs and FDC area in the small intestines of PP-deficient C57BL/Dk mice and immunocompetent C57BL/Dk (control) mice. For each mouse group, sections were stained with B-lymphocyte- (CD45R) and FDC- (CD21/35) specific antisera and the entire small intestine was examined microscopically. The number of ILFs was counted and the area occupied by FDCs was measured. (a) Intestines from PP-deficient mice contained significantly more (approximately four-fold more) ILFs than control mice. (b) Intestines from PP-deficient mice were found to have a significantly higher percentage of mILFs (46%) when compared to control mice (13%). (c) The area occupied by FDCs in individual mILFs from PP-deficient mice was significantly larger than those in mILFs from control mice. (d) The total area covered by FDCs in the mILFs in PP-deficient C57BL/Dk mice was equivalent to the total area covered by FDCs from all the PPs and mILFs in control mice. Each bar represents the mean ± SE for each group of mice (n = 12 for PP-deficient mice, n = 11 for control mice).

Figure 4

Enumeration of ILFs and FDC area in the small intestines of WT→WT mice, WT→LTα^–/– mice and WT→LTβ^–/– mice 105 days after reconstitution with WT bone marrow. For each mouse group, sections were stained with B-lymphocyte- (CD45R) and FDC- (CD21/35) specific antisera and the entire small intestine was examined microscopically. The number of ILFs was counted and the area occupied by FDCs was measured. (a) Similar numbers of ILFs were found in WT→WT mice, WT→LTa^–/– mice and WT→ LTβ^–/– mice. (b) However, WT→LTα^–/– and WT→LTβ^–/– mice were found to have a significantly higher percentage of mILFs when compared to WT→WT mice. (c) The area occupied by FDCs in individual mILFs from WT→LTα^–/– mice and WT→LTβ^–/– mice was significantly larger that those in WT→LTα^–/– mice. (d) The total area covered by FDCs in all the mILFs in WT→LTα^–/– mice or WT→LTβ^–/– mice was equivalent to the total area covered by FDCs in all the PPs and mILFs in WT→WT mice. Each bar represents the mean ± SE for each group of mice (n = 4 for each group).

Figure 5

Immunohistochemical detection of (a) GC B lymphocytes and (b) T lymphocytes within ILFs. Entire small intestines were examined from PP-deficient C57BL/Dk mice and representative images are shown. Sections were stained with B lymphocyte- (CD45R, green) and FDC- (CD21/35, red) specific antisera to distinguish between iILFs and mILFs (upper row). (a) To detect GC B lymphocytes, adjacent sections were stained with peanut agglutinin (green) (lower row). Peanut agglutinin-binding GC B lymphocytes were detected only in mILFs. (b) To detect T lymphocytes, adjacent sections were stained with CD3-specific monoclonal antiserum (green) (lower row). T lymphocytes were found in mILFs, whereas few or none were found in iILFs. Original magnification ×200.

Figure 6

The presence of mILFs in mice lacking PPs and MLNs correlates with enhanced IgA synthesis. Faecal IgA levels were determined by enzyme-linked immunosorbent assay and are expressed as the mean optical density (OD) at 450 nm for triplicate samples from individual mice. The horizontal bar represents mean OD for each experimental group of mice (n = 4). (a) In contrast to WT mice, faecal IgA levels in both LTα^–/– and LTβ^–/– mice were extremely low, but increased significantly after reconstitution with WT bone marrow. (b) C57BL/Dk (control) mice and PP-deficient C57BL/Dk mice were found to have equivalent levels of faecal IgA. SCID mice (+) were used as negative controls. *Indicates significant difference from WT mice (P = 0·05).