Ectopic LT alpha beta directs lymphoid organ neogenesis with concomitant expression of peripheral node addressin and a HEV-restricted sulfotransferase

Abstract

Lymph node (LN) function depends on T and B cell compartmentalization, antigen presenting cells, and high endothelial venules (HEVs) expressing mucosal addressin cell adhesion molecule (MAdCAM-1) and peripheral node addressin (PNAd), ligands for naive cell entrance into LNs. Luminal PNAd expression requires a HEV-restricted sulfotransferase (HEC-6ST). To investigate LT alpha beta's activities in lymphoid organogenesis, mice simultaneously expressing LT alpha and LT beta under rat insulin promoter II (RIP) control were compared with RIPLT alpha mice in a model of lymphoid neogenesis and with LT beta-/- mice. RIPLT alpha beta pancreata exhibited massive intra-islet mononuclear infiltrates that differed from the more sparse peri-islet cell accumulations in RIPLT alpha pancreata: separation into T and B cell areas was more distinct with prominent FDC networks, expression of lymphoid chemokines (CCL21, CCL19, and CXCL13) was more intense, and L-selectin+ cells were more frequent. In contrast to the predominant abluminal PNAd pattern of HEV in LT beta-/- MLN and RIPLT alpha pancreatic infiltrates, PNAd was expressed at the luminal and abluminal aspects of HEV in wild-type LN and in RIPLT alpha beta pancreata, coincident with HEC-6ST. These data highlight distinct roles of LT alpha and LT alpha beta in lymphoid organogenesis supporting the notion that HEC-6ST-dependent luminal PNAd is under regulation by LT alpha beta.

Figure 1.

LT expression in RIPLTβ and RIPLTαβ mice. In situ hybridization for RIPLTβ transgene transcription with a DIG-labeled antisense LTβ riboprobe on RIPLTβ pancreas (A) and kidney (B). In situ hybridization with DIG-labeled LTα (C and D) and LTβ (E and F) antisense riboprobes on RIPLTα (C) and RIPLTαβ (D–F) pancreas. E is a higher magnification of F showing the extensive cellular accumulation in RIPLTαβ pancreatic tissue. Objective 40× (A–E); objective 5× (F).

Figure 2.

Histologic comparison of C57BL/6 and RIPLT pancreata. Hematoxylin and eosin stain of pancreas of C57BL/6 (A), RIPLTα (B), RIPLTβ (C), and RIPLTαβ (D) mice. RIPLTβ pancreas is indistinguishable from that of nontransgenic mice. RIPLTα exhibit a peri-islet cellular accumulation in contrast to the extensive invasive intra-islet cellular accumulation observed in RIPLTαβ mice. Objective 20×.

Figure 3.

Cellular compartmentalization of RIPLTαβ infiltrates. RIPLTαβ pancreatic tissue were analyzed by immunohistochemistry to detect B cells, T cells, and FDCs within RIPLTαβ infiltrates. Serial sections (A–C) were stained with anti-B220 (A), anti-CD4 (B), and anti-CD8 (C). Serial sections (D and E) were stained with anti-B220 (D) and anti-CR1 (E). All were visualized with Vector Red substrate. Objective 40× (A–C); objective 20× (D and E). I, Islet tissue detected by anti-insulin staining (not shown).

Figure 4.

In situ hybridization analysis of lymphoid chemokine transcription in LN and RIPLTαβ pancreas. C57BL/6 PLN (A, C, and E) and serial sections of RIPLTαβ pancreas (B, D, and F) were probed with DIG-labeled antisense CCL21 (A and B), CCL19 (C and D), and CXCL13 (E and F) riboprobes. Positive signal is seen as dark purple staining. Arrows in A and B denote high magnification inset. I, Islet.

Figure 5.

LTαβ contributes to luminal PNAd expression on HEV. PNAd expression was detected by immunohistochemistry analysis with MECA 79 antibody in C57BL/6 PLN (A and B), RIPLTα pancreas (C and D), RIPLTαβ pancreas (E and F), and LTβ^−/− MLN (G and H). LTβ^−/− MLN exhibited a reduction in luminal MECA 79 expression. RIPLTαβ pancreata exhibited an increase in the number of MECA 79⁺ vessels and in luminal PNAd expression compared with the predominately abluminal pattern observed in RIPLTα pancreatic infiltrates. Objective 20× (A, C, E, and G); objective 40× (B, D, F, and H).

Figure 6.

LTαβ–induced luminal PNAd correlates with HEC-6ST expression. Fixed tissue sections of C57BL/6 PLN (top panel) and LTβ^−/− MLN (bottom panel) were analyzed by two-color immunofluorescence staining with MECA 79 (red) and anti-HEC-6ST (green) antibodies. C57BL/6 PLN exhibited pericellular PNAd expression with concomitant HEC-6ST expression in HEV. Top panel inset of C57BL/6 PLN-HEV demonstrates intracellular HEC-6ST expression and cell surface PNAd expression. Two-color analysis of LTβ^−/− MLN, with particular attention to vessels that show only abluminal MECA 79⁺ staining, reveals the absence of HEC-6ST expression. Objective 40×.

Figure 7.

RIPLTαβ-HEV show increased luminal PNAd and HEC-6ST expression. RIPLTα (top panel) and RIPLTαβ (bottom panel) pancreatic tissue sections were analyzed by two-color immunofluorescence analysis with MECA 79 (red) and anti-HEC-6ST (green) antibodies. The H&E stained image was merged with MECA 79 staining to orient the histologic location of the vessels; objective 20×. RIPLTα infiltrates exhibited predominately abluminal MECA 79⁺ staining vessels and no detectable HEC-6ST expression similar to LTβ^−/− MLN-HEV (Fig 6, bottom panel). In contrast, analysis of RIPLTαβ pancreata revealed both luminal and abluminal MECA 79⁺ vessels with coincident HEC-6ST expression on those luminal, pericellular MECA 79⁺ HEV. Bottom panel inset of RIPLTαβ HEV demonstrates intracellular HEC-6ST expression and cell surface PNAd expression–a pattern more reminiscent of C57BL/6 PLN (Fig 6, top panel) than RIPLTα infiltrates. Objective 40×.