Cloning of a cDNA Encoding Bovine Interleukin-18 and Analysis of IL-18 Expression in Macrophages and Its IFN-γ-Inducing Activity

Abstract

Interleukin-18 (IL-18) is a recently described cytokine that enhances interferon-gamma (IFN-gamma) production, either independently or synergistically with IL-12. These properties identify IL-18 as an immunoregulatory cytokine that may be pivotal in host defense against intracellular pathogens. We have isolated and sequenced a cDNA encoding bovine IL-18. The open reading frame (ORF) is 582 bp in length, encoding a predicted 192 amino acid (aa) precursor protein. Multiple sequence alignment demonstrated that bovine IL-18 has 65% and 78% identity with the predicted amino acid sequences of murine and human IL-18, respectively. IL-18 mRNA was constitutively present in bovine peripheral blood monocyte-derived macrophages (MDM), with no upregulation on stimulation with lipopolysaccharide (LPS). IL-18 transcripts were weakly detected in B lymphocytes but inducible in the B cell line BL-3. Human recombinant IL-18 (rHuIL-18) induced IFN-gamma production by PHA-stimulated peripheral blood mononuclear cells (PBMC), which was potentiated by rHuIL-12. Further, rHuIL-12 and rHuIL-18 enhanced proliferation of untreated PBMC. Antigen-specific T cell lines demonstrated IL-18-dependent enhancement of IFN-gamma production, indicating that bovine T cells are one of the leukocyte subsets that respond to IL-18. Analysis of IL-18 expression and its ability to induce IFN-gamma production by bovine lymphocytes are important considerations for understanding mechanisms of protective immunity and designing vaccines for intracellular pathogens.

DNA from Protozoan ParasitesBabesia bovis, Trypanosoma cruzi, andT. bruceiIs Mitogenic for B Lymphocytes and Stimulates Macrophage Expression of Interleukin-12, Tumor Necrosis Factor Alpha, and Nitric Oxide

“…Because the dot blot assay detects only p40 expression, a bioassay which measures the induction of IFN-␥ by bovine PBMC stimulated with functional heterodimeric IL-12 was also used to measure IL-12-like bioactivity (37,38). Supernatants from macrophages treated with E. coli, T. brucei, or B. bovis DNA induced significant IFN-␥ production by PBMC compared with those from macrophages treated with bovine DNA (Fig.…”

“…The presence of the biologically active IL-12 heterodimer was determined as described (37,38). Briefly, macrophage supernatants (200 l) were incubated with 2 ϫ 10 6 PBMC and phytohemagglutinin PHA (1 g/ml) in a total volume of 400 l in a 48-well plate for 2 days.…”

Interleukin-12 as an Adjuvant Promotes Immunoglobulin G and Type 1 Cytokine Recall Responses to Major Surface Protein 2 of the Ehrlichial PathogenAnaplasma marginale

“…An IL-12 bioassay based on induction of IFN-␥ by IL-12-and phytohemagglutinin (PHA)-stimulated PBMC was used (42). Diluted sera were incubated with 0.02 ng of IL-12 at 37°C for 1 h and then added along with 1 g of PHA (Sigma Chemical Co.) to 2 ϫ 10 6 PBMC cultured in 1 ml of complete medium.…”

Anaplasma marginale Major Surface Protein 2 CD4 + -T-Cell Epitopes Are Evenly Distributed in Conserved and Hypervariable Regions (HVR), Whereas Linear B-Cell Epitopes Are Predominantly Located in the HVR

“…When PBMC from all animals were stimulated with a mixture of phytohemagglutinin (PHA; 1.0 g per ml), IL-12 (0.01 ng per ml), and IL-18 (0.5 ng per ml), which served as a positive control for IFN-␥ synthesis (27,29), large numbers of IFN-␥-producing cells were detected, achieving or nearing saturation of the test wells. Antigens consisted of 10 and 1 g of uninfected bovine erythrocyte membranes (URBC) per ml, A. marginale Florida strain homogenate, native MSP2, or MSP2-derived peptides diluted in complete RPMI 1640 medium (11) ( ϩ T lymphocytes to Ͻ1% of total cells with CD4-specific MAb ILA-11A bound to magnetic beads (16) significantly reduced the number of lymphocytes responding to either A. marginale (P Ͻ 0.01) or MSP2 (P Ͻ 0.01) to numbers similar to background numbers from lymphocytes stimulated with medium alone or URBC (Fig.…”