Feline bone marrow derived macrophages : in vitro culturing technique, cloning and sequencing of feline TNF alpha and IL 1 beta cytokines, and potential modulation of macrophage gene expression by feline leukemia virus

Investigations of feline macrophage-viral interactions have been hampered due to difficulty in obtaining adequate macrophage numbers from tissues without euthanasia of the cat. We describe a method of small sample bone marrow culture which yields large numbers of macrophages (average 90% population) with residual lymphocytes and stromal cells, and does not require euthanasia. From cultured macrophages, cDNA for feline TNFα and IL-1β were cloned using degenerate primers and polymerase chain reaction technology. Sequenced feline TNFα cDNA matched 98.6% with coding regions for a genomic clone of feline TNFa, and 90%, 86%, 85%, 82% and 83% overall matches with cDNA for human, porcine, ovine, murine and goat TNFα, respectively. Full length TNFα cDNA was 702 bp with a predicted protein of 233 amino acids and a molecular weight of 25 Kd (precursor form of mature TNFα). Full length cDNA for feline IL-1β was 804 bp long with a predicted protein of 267 amino acids and a molecular weight of 31 Kd (precursor form). Sequenced feline IL-1β had 79%, 76%, 77% and 77% overall match with IL-1β cDNA for human, bovine, rabbit and murine species, respectively.

Bone marrow derived macrophages from normal and FeLV infected cats, were examined for differences in LPS induced TNFα or IL-1β mRNA expression. TNFα and Il-1β mRNA levels in macrophages from FeLV positive cats did not undergo modulation with one exception. For the exception, TNFα mRNA expression was not LPS induced and Il-1β mRNA level was significantly decreased. Viral antigen was detected in macrophages from this cat, indicating viral replication was occurring within some macrophages. No viral antigen was detectable in macrophages of other FeLV infected cats, virus was present only in residual lymphocytes. These findings suggest FeLV may modulate TNFα and IL-1β gene expression, but only when macrophages are actively replicating viruses. However, one must be cautious in interpretation of these results, since macrophages derived from the bone marrow of random source cats (known to be FeLV positive and FIV negative), may contain other undetected factors which contribute to modulation of gene expression.