Pulmonary expression of CXC chemokine ligand 13, CC chemokine ligand 19, and CC chemokine ligand 21 is essential for local immunity to influenza

Abstract

CXC chemokine ligand 13 (CXCL13), CC chemokine ligand 21 (CCL21), and CCL19 are constitutively expressed in secondary lymphoid organs, where they control the placement of lymphocytes and dendritic cells. However, these chemokines are also inducibly expressed in the lung after influenza infection. Here we show that, in the absence of spleen and lymph nodes, the expression of homeostatic chemokines in the lung is essential for local B and T cell responses to influenza and for the development and organization of inducible bronchus-associated lymphoid tissue (iBALT). Surprisingly, despite the association between local CXCL13 expression and the formation of ectopic lymphoid tissues, the loss of CXCL13 in the lung had minimal impact on either the development or function of iBALT. In contrast, the loss of CCL19 and CCL21 impaired iBALT formation as well as B and T cell responses. These results demonstrate that the local expression of homeostatic chemokines in nonlymphoid organs, such as the lung, plays an important role in protective immune responses.

Fig. 1.

Influenza-specific CD8 responses are impaired in spleen- and LN-deficient plt/plt mice. (A and B) Spleen- and LN-deficient mice were infected with influenza, and the frequencies of CD8 T cells (A) and NP-specific CD8 T cells (B) in the lungs were evaluated by flow cytometry. The data shown are gated on live lymphocytes (A) or live CD8 T cells (B). (C–E) Total leukocytes were enumerated in the lungs of infected mice by counting (C), and the total numbers of CD8 T cells (D) and NP-specific CD8 T cells (E) were calculated. There were four to five mice per group at each time. Statistical significance was evaluated by using an unpaired t test. (F and G) The frequency of activated DCs in the lungs was determined 7 days after infection by flow cytometry (F), and the total number of CD11c⁺IA^b+ cells in the lungs was calculated (G). (H) Serial dilutions of total leukocytes from the lungs of infected mice were cultured overnight with NP-specific T cell hybridoma cells that contained an IL-2–β-galactosidase reporter construct, and blue cells were enumerated.

Fig. 2.

The formation of germinal centers and the production of antibodies are impaired in spleen- and LN-deficient plt/plt mice. (A and B) Spleen- and LN-deficient mice were infected with influenza, and the frequencies of CD19 B cells (A) and GCB (B) in the lungs were evaluated by flow cytometry. PNA, peanut agglutinin. The data shown are gated on live lymphocytes (A) or live CD19 B cells (B). (C and D) The total numbers of CD19 B cells (C) or GCB (D) in the lungs of infected mice were calculated. (E) Serum titers of influenza-specific IgM and IgG were determined by ELISA. Flow cytometry was performed by using four to five mice per group, and antibody titers were determined in 7 to 10 mice per group. Statistical significance was evaluated by using an unpaired t test.

Fig. 3.

Prolonged morbidity and a delay in viral clearance correlate with the inability to express homeostatic chemokines in the lung. (A) Spleen- and LN-deficient mice were infected with influenza and weighed daily. (B) Viral titers in the lungs were assayed in embryonated eggs.

Fig. 4.

The formation of iBALT requires both CXCL13 and CCL19/CCL21. (A and B) Spleen- and LN-deficient mice were infected with influenza, and frozen sections of lungs were stained with MacNeal's tetrachrome. (C–E) Sections were probed with anti-B220 and anti-Thy-1.2 to visualize B and T cells (C), with anti-B220 and anti-CD21 to show B cells and FDCs (D), or with anti-ERTR7 and anti-BP3 to identify stromal cells (E). (Magnification: A and B, ×25; C–E, ×100.)

Fig. 5.

The expression of homeostatic chemokines in iBALT. Spleen- and LN-deficient mice were infected with influenza. (A and B) Frozen sections of infected lungs were probed with anti-CCL21 and anti-PNAd to visualize HEVs (A) and with anti-CXCL13 to visualize B cell follicles (B). (Magnification: ×200.) (C) RNA was extracted from frozen sections that contained areas of iBALT, and quantitative PCR was performed to evaluate the expression of LTβ, CXCL12, CXCL13, CCL19, CCL21, and AID. Expression of mRNA was normalized to the levels of mRNA in C57BL/6 mice on day 10, which was set at 1. Reciprocal BM chimeras were generated by using C57BL/6 and Cxcl13^−/− mice as well as C57BL/6 and plt/plt mice. (D) Mice were allowed to reconstitute for 8 weeks and were infected with influenza. RNA was extracted from the lungs and the expression of CXCL13, CCL21, and CCL19 was assayed by quantitative PCR.