Purification and Partial Amino Acid Sequence of Rabbit Tumor Necrosis Factor

Abstract

Good production of tumor necrosis factor (TNF) in the rabbit was obtained using Propionibacterium acnes IID 912 as a priming agent and subsequent administration of lipopolysaccharide. The physicochemical characteristics of rabbit TNF were very similar to those of murine TNF. The molecular weight of rabbit TNF was 39,000 as estimated by gel filtration, and 18,000 by SDS-PAGE. The isoelectric point was determined as pH 4.0 by isoelectric focusing. Rabbit TNF was stable within the pH range of 5.5 to 11.0, and was stable at 56 degrees C for 8 hr. It was digested by trypsin, pancreatic protease and elastase, but was resistant to neuraminidase. The amino acid sequence of rabbit TNF was determined as Ser-Ala-Ser-Arg-Ala-Leu- .... Monoclonal antibody against rabbit TNF completely inhibited both the in vivo and in vitro activity of rabbit TNF. However, this antibody could not inhibit the action of murine TNF. Antitumor activity of rabbit TNF was shown against murine and human cancer cells in vivo and in vitro, and rabbit TNF was also capable of distinguishing malignant cells from normal cells.

Evaluation of recombinant human tumor necrosis factor by scheduled intratumoral administration in mice bearing transplantable tumors.

“…TNF shows in vitro cytotoxic activity to a wide variety of human and animal malignant cell including cell lines and fresh cells of cancer patients in primary culture (Carswell et al 1975 ;Ha et al 1985 ;Haranaka et al 1985 ;Watanabe et al 1985). However, rTNF at the concentrations of 1 to 1,000 units/ml did not show any direct cytotoxicity to Meth A and Sarcoma-180 in primary culture (our unpublished results), indicating that the necrotic effect and the tumor regression are not direct action by rTNF.…”

“…However, rTNF at the concentrations of 1 to 1,000 units/ml did not show any direct cytotoxicity to Meth A and Sarcoma-180 in primary culture (our unpublished results), indicating that the necrotic effect and the tumor regression are not direct action by rTNF. Several groups reported that TNF shows no toxicity to cultured Meth A (Carswell et al 1975 ;Ruff and Gifford 1981;Sugarman et al 1985;Yoshie et al 1986) except 3 groups (Ha et al 1985;Haranaka et al 1985 ;Kull et al 1985), although Meth A has TNF receptors (Kull et al 1985;Yoshie et al 1986). There is no correlation between the number of TNF receptors on the cell surface and expression of the cytotoxicity (Kull et al 1985;Yoshie et al 1986).…”

“…Tumor necrosis factor (TNF), a cytokine released by activated monocytes and/or macrophages, shows in vitro cytotoxicity to a wide variety of human and animal malignant cells including cell lines such as mouse L cells, P388, HL-60, ML-1, HeLa, and Raji and fresh cells of cancer patients in primary culture (Carswell et al 1975; Ha et al 1985; Haranaka et al 1985; Kull et al 1985; Watanabe et al 1985; Yoshie et al 1986) and causes hemorrhagic necrosis followed by tumor regression in transplanted tumors in mice (Matthews 1981(Matthews , 1983; Fisch and Gifford 1983; Kelker et al 1985). Human TNF has been purified and the cDNA was cloned and expressed in Escherichia coli in several laboratories (Pennica et al 1984; Marmenout et al 1985; Shirai et al 1985).…”

Functional Distinction between CXC Chemokines, Interleukin-8 (IL-8), and Growth Related Oncogene (GRO)α in Neutrophil Infiltration

“…Neutralizing anti-rabbit IL-8 IgG and anti-rabbit GRO␣ IgG were raised in a goat, and purified with affinity column coupled with each chemokine (Matsukawa et al, 1997. Neutralizing anti-rabbit TNF␣ mAb was kindly provided by Dr. H. Nariuchi (The University of Tokyo, Japan) (Haranaka et al, 1985). Neutralizing anti-rabbit GRO␣ mAb (IgG1) was developed as described previously .…”

IL-8 is an essential mediator of the increased delayed-phase vascular permeability in LPS-induced rabbit pleurisy

“…Endotoxin content was less than 0.9 ng/mg protein. Neutralizing anti-TNF-␣ mouse monoclonal antibody (IgG1) was purified on a HiTrap Protein G column (Pharmacia LKB Biotechnology, Uppsala, Sweden) from ascites of BALB/c mice bearing hybridoma 2-11E (a generous gift from Dr. H. Nariuchi, The University of Tokyo, Japan) [14]. Endotoxin content was less than 3.2 ng/mg protein.…”