Molecular cloning and characterization of porcine innate immunoreceptor complexes in natural killer cells and phagocytes

Use of pigs as an animal model system has a great potential for immunological research, such as development of innate immune system. This study describes molecular cloning and characterization of porcine immunoreceptor complexes MDL-1/DAP12 and NKG2D/DAP10. These receptors are involved in cell activation of innate immune system. Porcine DAP12 and DAP10 genes were cloned from PBL cDNA library. DAP12 contains an ITAM, and DAP10 contains a PI-3 kinase binding site. Porcine DAP12 and DAP10 genes are located on chromosome 6q21. They are genetically linked in opposite transcriptional orientation. In Northern blot analysis, DAP12 and DAP10 are predominantly expressed in lympho-hematopoietic cells. Myeloid DAP12-associating lectin-1 (MDL-1) is a type II membrane protein that associates with DAP12. Two isoforms of porcine MDL-1 cDNA were cloned from pulmonary alveolar macrophages. MDL-1 transcripts were detected mainly in macrophages and monocytes. MDL-1 is expressed on the cell surface associated with DAP12. Human CD94 alone cannot be associated with DAP12 without NKG2C whereas mouse CD94 directly associates with DAP12. A mutation of the lysine residue in the mouse CD94 transmembrane region abolished the association between mouse CD94 and DAP12. A difference in the association pattern of CD94/NKG2C with DAP12 is found in rodent and other mammals. NKG2D is a C-type lectin family receptor that associates with DAP10. Porcine NKG2D was cloned by RT-PCR. Porcine NKG2D cDNA has an open reading frame of 642 bp. Its expected polypeptide sequence is 214 amino acids. Porcine NKG2D has 66% sequence identity with human NKG2D. Porcine NKG2D requires DAP10 for cell surface expression. Porcine CD69 cDNA contains 600 bp open reading frame. Porcine CD69 has 67% sequence identity with human CD69. Porcine CD69 mRNA was detected in bone marrow, spleen, thymus, and lymph nodes, but not in liver, muscle, and mammary gland. CD69 mRNA was detected in activated PBL, NK cells, macrophages, and granulocytes, but not in resting cells. Thus, CD69 can be used as an activation marker in porcine immune cells. NKG2D and CD69 genes are located on chromosome 5q25, indicating that chromosome 5q25 contains pig NK gene complex.