Alymphoplasia (aly)-type nuclear factor kappaB-inducing kinase (NIK) causes defects in secondary lymphoid tissue chemokine receptor signaling and homing of peritoneal cells to the gut-associated lymphatic tissue system

Abstract

Alymphoplasia (aly) mice, which carry a point mutation in the nuclear factor kappaB-inducing kinase (NIK) gene, are characterized by the systemic absence of lymph nodes and Peyer's patches, disorganized splenic and thymic architectures, and immunodeficiency. Another unique feature of aly/aly mice is that their peritoneal cavity contains more B1 cells than normal and aly/+ mice. Transfer experiments of peritoneal lymphocytes from aly/aly mice into recombination activating gene (RAG)-2(-/-) mice revealed that B and T cells fail to migrate to other lymphoid tissues, particularly to the gut-associated lymphatic tissue system. In vivo homing defects of aly/aly peritoneal cells correlated with reduction of their in vitro chemotactic responses to secondary lymphoid tissue chemokine (SLC) and B lymphocyte chemoattractant (BLC). The migration defect of aly/aly lymphocytes was not due to a lack of expression of chemokines and their receptors, but rather to impaired signal transduction downstream of the receptors for SLC, indicating that NIK is involved in the chemokine signaling pathway known to couple only with G proteins. The results showed that the reduced serum levels of immunoglobulins (Igs) and the absence of class switch to IgA in aly/aly mice are due, at least in part, to a migration defect of lymphocytes to the proper microenvironment where B cells proliferate and differentiate into Ig-producing cells.

Figure 1

Abundant B1 cells in the PEC and complete absence of B cell populations in LP of aly/aly mice. (A) Flow cytometric analysis of PEC cells from aly/aly and aly/+ mice. A total of 2 × 10⁴ cells were analyzed for IgM in combination with Mac-1. The numbers are the percentages of B1 (IgM⁺Mac-1⁺) and B2 (IgM⁺Mac-1⁻) cells. (B) Total numbers of PEC cells in aly/aly and aly/+ mice, as calculated by (percentage of indicated cells) × (number of viable cells). (C and D) Flow cytometric analysis of LP cells of aly/aly and aly/+ mice, stained with FITC-conjugated anti-B220 and PE-conjugated anti-IgM or anti-IgA. The numbers are the percentages of B cells (B220⁺IgM⁺) and plasma cells (B220⁻IgA⁺).

Figure 2

Transfer of PEC cells from aly/aly mice fails to generate IgA plasma cells in GALT of RAG-2^−/− mice. Flow cytometric analysis of LP cells (A) and MLNs (B) derived from RAG-2^−/− mice, RAG-2^−/− mice injected with 1.3 × 10⁷ PEC cells from aly/aly and aly/+ mice, and 3 × 10⁷ PEC cells from LTα^−/− mice, at 6 and 12 wk, respectively, after the transfer. A total of 2 × 10⁴ cells were analyzed for B220 in combination with IgA. The numbers are the percentages of IgA plasma cells (B220⁻IgA⁺). (C) RAG-2^−/− mice, RAG- 2^−/− mice that were injected intraperitoneally with PEC cells from aly/aly and aly/+ mice 3 wk previously, and RAG-2^−/− mice at 12 wk after injection with LTα^−/− PEC cells. A total of 2 × 10⁴ cells were analyzed for IgM in combination with Mac-1. The numbers are the percentages of B1 (IgM⁺Mac-1⁺) and B2 (IgM ⁺Mac-1⁻) cells.

Figure 3

Isotype of serum Ig in RAG-2^−/− mice injected with PEC cells from aly/aly (▪) and aly/+ (○) mice. Serum Ig levels were determined 3 or 6 wk after transfer, by isotype-specific ELISA. Results represent mean ± SE from three separate experiments.

Figure 4

Migration defect of aly/aly peritoneal T cells to the GALT system. (A) Flow cytometric analysis of LP cells derived from RAG-2^−/− mice and RAG-2^−/− mice transferred with 1.3 × 10⁷ PEC cells from aly/aly and aly/+ mice 6 wk previously. A total of 2 × 10⁴ cells were analyzed for CD3 in combination with Mac-1. The numbers are the percentages of T (CD3⁺Mac-1⁻) cells. (B) Numbers of T cells recovered 3–6 wk after transfer, calculated by multiplying total cell number with the percentage of T cells in indicated lymphoid tissues. Results represent mean ± SE from three different experiments.

Figure 5

Chemokine and chemokine receptor expression in aly/aly mice. (A) Chemokine expression in spleen and small intestine of RAG-2^−/−, aly/aly, and aly/+ mice. Northern blot analysis of total RNA from spleen and small intestine of RAG-2^−/− mice and aly/aly mice, and small intestine with or without PPs from aly/+ mice, and probed to detect expression of SLC, ELC, and BLC. (B) Chemokine receptor expression in spleen and PEC cells from aly/aly and aly/+ mice. LPS stimulation markedly increased the expression of CCR7 and BLR1 RNA in peritoneal cells of aly/+ but not aly/aly mice. Northern blot analysis of total RNA from unstimulated and LPS-stimulated (20 μg of LPS for 2 h) splenic and PEC cells and probed to detect expression of CCR7 and BLR1. GAPDH hybridization indicates amount of total RNA loaded in each lane.

Figure 6

Decreased chemotactic activity of SLC and BLC on resting and acutely activated PEC cells from aly/aly mice compared with aly/+ mice. The number of input and migrating cells of each subtype was determined by immunostaining and flow cytometry. Results are expressed as the percentage of input cells of each subtype migrating to the lower chamber of a Transwell filter. Graphs show migration of: (A) PEC B1 cells; (B) PEC B2 cells; (C) PEC T cells; (D) spleen B cells; (E) spleen T cells, towards SLC; (F) PEC B1 cells; (G) PEC B2 cells; and (H) spleen B cells towards BLC. PEC cells and spleen cells were preincubated with medium alone (LPS−) or LPS at 20 μg/ml for 3 h. Data points represent the mean ± SE for experiments performed in triplicate. Each experiment was repeated at least three times.

Figure 7

Impaired activation of NF-κB by SLC in aly/aly PEC cells. Nuclear proteins (6 μg) from unstimulated and SLC (500 ng/ml) stimulated PEC cells for 15 or 30 min, were incubated with unlabeled (cold competitor, WT comp) and mutant (Mut comp) NF-κB probe for 20 min. The samples were then incubated with ³²P-labeled NF-κB sequence for 20 min, followed by separation on polyacrylamide gel and analysis by autoradiography. The Oct-1–specific probe was used as internal control.

Figure 8

The NF-κB complexes are different in PEC cells from aly/aly and aly/+ mice. Nuclear extracts from PEC cells stimulated for 30 min with SLC were incubated with the indicated antibody followed by ³²P-labeled NF-κB sequence. The samples were migrated on polyacrylamide gels and analyzed by autoradiography. (A) p65-cRel/p65-RelB complex, (B) p50-p65/p50-RelB complex, and (C) p50-p50 complexes.