Critical roles for IFN-beta in lymphoid development, myelopoiesis, and tumor development: links to tumor necrosis factor alpha

Abstract

We have generated mice null for IFN-beta and report the diverse consequences of IFN-beta for both the innate and adaptive arms of immunity. Despite no abnormalities in the proportional balance of CD4 and CD8 T cell populations in the peripheral blood, thymus, and spleen of IFN-beta-/- mice, activated lymph node and splenic T lymphocytes exhibit enhanced T cell proliferation and decreased tumor necrosis factor alpha production, relative to IFN-beta+/+ mice. Notably, constitutive and induced expression of tumor necrosis factor alpha is reduced in the spleen and bone marrow (BM) macrophages, respectively, of IFN-beta-/- mice. We also observe an altered splenic architecture in IFN-beta-/- mice and a reduction in resident macrophages. We identify a potential defect in B cell maturation in IFN-beta-/- mice, associated with a decrease in B220+ve/high/CD43-ve BM-derived cells and a reduction in BP-1, IgM, and CD23 expression. Circulating IgM-, Mac-1-, and Gr-1-positive cells are also substantially decreased in IFN-beta-/- mice. The decrease in the numbers of circulating macrophages and granulocytes likely reflects defective maturation of primitive BM hematopoiesis in mice, shown by the reduction of colony-forming units, granulocyte-macrophage. We proceeded to evaluate the in vivo growth of malignant cells in the IFN-beta-/- background and give evidence that Lewis lung carcinoma-specific tumor growth is more aggressive in IFN-beta-/- mice. Taken altogether, our data suggest that, in addition to the direct growth-inhibitory effects on tumor cells, IFN-beta is required during different stages of maturation in the development of the immune system.

Fig. 1.

Defect in BM B cell maturation in IFN-β^-/- mice. BM cells, harvested from IFN-β^+/+ and IFN-β^-/- mice, were depleted of RBC and analyzed by flow cytometry after labeling with the indicated Abs. (A) B220- and CD43-lineage cells. B220-positive cells are shown subdivided into fractions I (B220^+ve/CD43^+ve), II (B220^+ve/low/CD43^-ve), and III (B220^+ve/high/CD43^-ve). (B-D) B220 and BP-1, IgM, and CD23 lineages. Where indicated, numbers represent the gated cell population as a percentage of total cell population. Data represent the mean from groups of five mice. ^*, P < 0.05; ^**, P < 0.01; ^***, P < 0.001. +/+, IFN-β^+/+; -/-, IFN-β^-/-.

Fig. 2.

Defective maturation of CFU-GM hematopoietic progenitors in the BM of IFN-β^-/- mice. BM mononuclear cells from IFN-β^+/+ and IFN-β^-/- mice were plated in a methylcellulose assay system. The data are expressed as the mean total number of cfu ± SEM from three independent experiments. CFU-GM, burst-forming units (BFU-E), and mixed-lineage colonies (CFUGEMM). ^*, P = 0.049; ^**, P = 0.01.

Fig. 3.

Impaired LPS-induced TNF-α production from IFN-β^-/- BMMs. BMMs from IFN-β^+/+ and IFN-β^-/- mice were cultured for 24 h after LPS stimulation (10 and 100 ng/ml), in the presence or absence of rIFN-β. Supernatants were assayed for TNF-α in triplicate by ELISA. Data are representative of the mean ± SEM of two independent assays. (IFN-β^+/+ n = 5; IFN-β^-/- n = 5). ND, not detected; ^***, P ≤ 0.0001.

Fig. 4.

Comparative analysis of splenic tissues from naïve IFN-β^+/+ and IFN-β^-/- mice. Paraffin-embedded spleen sections were stained with anti-B220 (brown) and anti-CD3 (blue) (A-D), anti-TNF-α (red) (E and F), or anti-Mac-3 (brown) (G and H) Abs. wp, white pulp; rp, red pulp; b, B cell follicles; t, PALS; ca, central arterioles. +/+, IFN-β^+/+; -/-, IFN-β^-/-. (Original magnification: A, B, and E-H, ×10; C and D, ×40.)

Fig. 5.

Enhanced T cell proliferation in the absence of IFN-β. Lymph node cells from IFN-β^+/+ (○) and IFN-β^-/- (•) mice were stimulated with murine recombinant anti-CD3 (3 μg/ml)/anti-CD28 (1 μg/ml), in the presence (B-D)or absence (A) of murine rIFN-β (10, 100, or 1,000 units/ml) then pulsed for 18 h with [methyl-³H]thymidine. [³H]Thymidine uptake was measured over 96 h. Data are representative of the mean ± SEM from three independent experiments (P = 0.002).

Fig. 6.

Reduced levels of TNF-α in activated IFN-β^-/- T cells. Splenocytes were stimulated with mouse recombinant anti-CD3 for 48 h, followed by PMA and ionomycin for 4 h. Cells were then simultaneously labeled with cell-surface Abs for CD4 (A-D) or CD8 (E-H) and stained for either a nonspecific antibody with the same isotype (A and E) or intracellular cytokine antibodies as indicated. Values shown are the lymphocyte population as a percentage of total splenic population. Numbers in parentheses represent the percentage of either CD4- or CD8-positive cytokine-secreting cells. Statistical analysis was by Student's t test; ^**, P = 0.008; ^***, P = 0.001. +/+, IFN-β^+/+; -/-, IFN-β^-/-. Data are representative from IFN-β^+/+ (n = 5) and IFN-β^-/- (n = 5) mice.

Fig. 7.

Impaired anti-LLC-1 tumor response in IFN-β^-/- mice. IFN-β^+/+ (○) and IFN-β^-/- (•) mice were inoculated s.c. with 10⁶ LLC-1 cells and monitored daily for tumor growth, as indicated. Each point is representative of the average tumor size for the group ± SEM. ^*, P ≤ 0.05; ^**, P ≤ 0.01.