Molecular cloning and physiological effects of brushtail possum interleukin-1beta.
Interleukin-1beta (IL-1beta) was isolated from LPS-stimulated brushtail possum alveolar macrophages using PCR primers based on conserved regions of mammalian IL-1beta. The complete cDNA was cloned by 5' and 3' rapid amplification of cDNA ends (RACE). The predicted protein of 269 amino acids shared 4346% identity with several mammalian IL-1beta proteins. Constructs were made to express the mature IL-1beta in Escherichia coli and two recombinant IL-1beta proteins, rpIL-1beta1 and rpIL-1beta2, which differed in length by four amino acids at the N-terminus, were produced. Both proteins induced a weak proliferative response in a possum thymocyte assay. Possums injected intravenously with 100 microg of rpIL-1beta1 or rpIL-1beta2 showed profound changes in body temperature and numbers of circulating leukocytes. A sharp decrease in temperature occurred within 2 h of administration followed by an elevation of temperature peaking at 24 h. The smaller rpIL-1beta1 protein had a greater effect on temperature than rpIL-1beta2. Both rpIL-1beta proteins caused a marked decrease in number of neutrophils and lymphocytes at 2-6 h after injection. At 24 h after injection, neutrophil and lymphocyte numbers were elevated 6.0-fold and 2.6-fold, respectively in the possums injected with rpIL-1beta1 and 3.9-fold and 1.5-fold, respectively in the possums injected with rpIL-1beta2. Fibrinogen levels were elevated at 24 and 72 h after injection with both proteins. In comparison, neither recombinant bovine IL-1beta (rbIL-1beta) nor PBS had significant effects on body temperature or blood haematology. The studies have shown that the two recombinant forms of IL-1beta were biologically active in possums and that the IL-1beta with four fewer amino acids at the N-terminus was the more active.
Wedlock
D N
DN
AgResearch, Wallaceville Animal Research Centre, Upper Hutt, New Zealand. wedlockn@agresearch.cri.nz
Goh
L P
LP
Parlane
N A
NA
Buddle
B M
BM
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002417:Cattle; D003001:Cloning, Molecular; D006801:Humans; D007275:Injections, Intravenous; D007375:Interleukin-1 / Q000235:genetics* / Q000502:physiology*; D051379:Mice; D008969:Molecular Sequence Data; D009893:Opossums / Q000235:genetics* / Q000502:physiology*; D011817:Rabbits; D011994:Recombinant Proteins / Q000008:administration & dosage / Q000096:biosynthesis / Q000494:pharmacology
D007375:Interleukin-1; D011994:Recombinant Proteins