Comparison of vasoactive intestinal peptide-mediated protein phosphorylation in human lymphoblasts and colonic epithelial cells.

Vasoactive intestinal peptide (VIP) induces phosphorylation of a basic 38,000 mol. wt protein in a human lymphoblastic cell line (Molt 4b) and a human colon carcinoma cell line (HT29). In both cell types, VIP interacts with specific high affinity receptors to activate adenylate cyclase and cAMP-dependent protein kinase. The two cell types appear to express homologous receptors with similar affinity and specificity for VIP, but the colonic epithelial cells express a greater number of receptors. HT29 colonic cells also exhibit a greater stimulation of adenylate cyclase and a higher phosphorylation index for the 38,000 mol. wt protein in response to VIP. This 38,000 mol. wt protein, which is phosphorylated in the presence of VIP, appears to be identical in both cell lines; it is phosphorylated in both lymphoblasts and colonic epithelial cells in the presence of forskolin, but not in the presence of phorbol 12-myristate 13-acetate. Phosphorylation of this 38,000 mol. wt protein may be an important step in VIP regulation of water and electrolyte secretion from colonic epithelial cells, and in VIP regulation of immunoglobulin and lymphokine secretion from lymphocytes.

Identification of high affinity receptors for vasoactive intestinal peptide on human lymphocytes of B cell lineage.

Specific, high affinity receptors for vasoactive intestinal peptide (VIP) have been identified on a human pre-B cell line, Nalm 6, and on a human plasma cell line, Dakiki. The single class of high affinity sites exhibited a KD of 12.6 +/- 2.9 nM for VIP in Nalm 6 cells and 9.1 +/- 2.7 nM in Dakiki plasma cells. The homologous peptides, peptide histidine methionine (PHM), growth hormone releasing factor (GHRF), and secretin were all less effective than VIP in competitively inhibiting binding of 125I-VIP to Nalm 6 and Dakiki plasma membranes. The putative receptor was characterized as a 47-kDa protein using covalent cross-linking techniques and VIP stimulated adenylate cyclase in pre-B cells. Human lymphocytes of B cell lineage thus appear to express functional VIP receptors homologous to the receptor identified in T lymphoblasts, brain, pituitary, and intestine.

Alveolar macrophage subpopulations identified by differential centrifugation on a discontinuous albumin density gradient.

Mononuclear phagocytes are functionally heterogeneous. To study the relationship of the heterogeneous populations of macrophages from the lung, alveolar macrophages from Syrian hamsters that had been immunized and rechallenged intratracheally with Mycobacterium bovis (Strain BCG) were separated by discontinuous albumin gradient centrifugation into 5 subpopulations designated A, B, C, D, and E. An activated alveolar macrophage subpopulation (defined by the ability to destroy tumor target cells) was enriched only in fraction D. Cells in fraction D destroyed 40% of the tumor cells, whereas unseparated alveolar macrophages destroyed 29%. Fractions A, B, C, and E destroyed less than 15% of the tumor cells. The subpopulations were functionally heterogeneous with regard to chemotactic responsiveness, Fc receptor activity, and phagocytic activity. Incubation of the subpopulations with a lymphocyte supernatant from spleen cells stimulated with concanavalin A enhanced the cytotoxic activity of fraction D and activated cells in fractions C and E to destroy tumor cells. Neither resident alveolar macrophages nor any of the subpopulations destroyed tumor cells. Only resident fraction D cells killed tumor cells when incubated with lymphokine containing supernatant fluids. The results are consistent with the hypothesis that some subpopulations of alveolar macrophages may be related and exist as a continuum of differentiation.

The effects of lipopolysaccharide, BCG-immune T lymphocytes, and lymphokines on generations of tumoricidal pulmonary macrophages in Syrian hamster.

Bacterial lipopolysaccharide (LPS) stimulates pulmonary macrophages from BCG immune-rechallenged hamsters to kill tumor cells in vitro. However, pulmonary macrophages from BCG immune and from untreated hamsters cannot be activated for tumor cytotoxicity by in vitro treatment with LPS. Pulmonary macrophages from the nonimmune hamsters acquire tumoricidal capacity after 3 hr of coculture with T cells from BCG immune-rechallenged hamsters or when incubated with Con-A-stimulated spleen cell supernatant fluid. A heterogeneous population of pulmonary lavage cells from BCG immune and from BCG immune-rechallenged hamsters destroys the tumor cells more effectively than a homogeneous population of pulmonary macrophages from the same animals. LPS significantly augments the cytotoxic activity of the heterogeneous population of pulmonary lavage cells.

Lack of lipopolysaccharide response of inbred strains of Syrian hamsters.

A variety of culture conditions were used to assess the lipopolysaccharide (LPS) response of spleen cells from several strains of Syrian hamsters. The cells failed to respond to LPS while good responses were obtained with Concanavalin A and poke week mitogen. Stimulation of macrophages with LPS failed to induce tumor cell destruction unless the macrophages were primed by immunization with BCG. The results indicated that hamsters are unresponsive to LPS.

Synthesis and antimetastatic properties of steroeoisomeric trycylic bis(dioxopiperazine) analogues in a B16 melanoma model.

The synthesis for trans and cis tricyclic bis(dioxopiperazine)s 5 and 6 from pyrazine-2,3-dicarboxamide (7) is described. Stereoselective antimetastatic activity differences for these analogues were observed following pretreatment of B16-F10 melanoma cells in vitro. Activities for these isomers were compared with selected intermediates, and the data are discussed in relation to previous results obtained with cis- and trans-cyclopropane analogues.

Effect of stereoisomers related to ICRF-159 on metastasis of B16 melanoma.

The antitumour effects of ICRF-159 and related analogues were evaluated using the B16 melanoma. Treatment of mice with ICRF-159 inhibited tumour growth, while each of the analogues, trans-4,4(1)-(1,2-cyclopropandiyl) bis (2,6-piperazinedione) (trans-5), and cis-4,4(1)-(1,2-cyclopropandiyl) bis (2,6-piperazinedione) (cis-7) independently accelerated primary tumour growth. Pretreatment of B16 melanoma cultures either with ICRF-159 or the analogue cis-7 decreased the yield of lung-colonies following i.v. injection of tumour cells. In contrast, pretreatment of tumour cells with the trans-5 analogue led to an increase in lung colonies. The effect on colony formation in vitro of these analogues correlated with increased growth in vivo, and not with lung colony formation.

Destruction of tumor cells by BCG-activated alqolar macrophages.

Alveolar macrophages obtained from Syrian golden hamsters were tested for their ability to destroy tumor cells. Only macrophages obtained from BCG immune animals rechallenged intratracheally with BCG five days before assay exhibited cytotoxic activity. Maximum destruction of tumor cells occurred after 5 days of incubation. Immunologic activation of macrophages was required to attain cytotoxic alveolar macrophages. Induction of inflammatory lung exudates by a variety of nonspecific irritants did not result in tumor cell destruction by macrophages. These observations may prove useful in designing an approach for immunotherapy of lung cancer.