Generation of procoagulant activity by mononuclear phagocytes: optimisation of an in vitro assay.

Leucocyte procoagulant activity (PCA) has previously been advocated as a useful measurement of cell-mediated immune reactivity. The assay is, however, susceptible to inter- and intra-experimental variation. This investigation identifies several factors which influence the stability and reproducibility of the test. Major factors which affect the recalcification time of plasma, include plasma lability, medium/buffer pH and both the nature and concentration of the indicator cells used in the assay. C. parvum-induced mouse peritoneal exudate cells have been used as a novel source of mononuclear phagocytes in the generation of PCA. Their sensitivity as indicator cells has been demonstrated by their responsiveness to stimulation by phytomitogen, endotoxin, and lymphokine (macrophage procoagulant-inducing factor). A simple test based on antigen-induced PCA of these cells, has provided an in vitro index of in vivo sensitisation to sheep red blood cells. Optimisation and standardisation of conditions detailed in this report for estimating PCA, renders the assay of value in monitoring lymphocyte and macrophage activation.

Serum-induced proliferation of retinal capillary endothelial cells is inhibited in the presence of retinal extract.

Both crude retinal extract and serum contain growth factors for retinal capillary endothelial cells (EC). This study, however, has shown that the combined presence of both crude retinal extract (bovine) and adult serum, induces profound inhibition of EC growth in vitro. EC grown under these conditions develop as sparse colonies of cells which appear to be inhibited from spreading on the fibronectin-coated surface. The extract/serum mixture is not toxic to the cells since it does not affect the viability and typical cobblestone morphology of established confluent cultures. Combinations of retinal extract with adult sera from various species exert the inhibitory effect but foetal or newborn serum is not affected by the extract. Growth inhibition is also found with crude bovine brain extract but cannot be ascribed to the presence of basic fibroblast growth factor (bFGF) or heparin in the extract, nor to a previously identified heat-labile (56 degrees C, 30 min) inhibitory factor in retinal extract. The inhibition is selective for endothelial cells in that mural cells (pericytes) remain unaffected. The nature and relevance of this inhibitory activity remains to be determined.

Retinal capillary endothelial cells prefer different substrates for growth and migration.

Recent studies have shown that the extracellular matrix modifies the behaviour of endothelial cells. We have studied the effects of extracellular matrix components on retinal capillary endothelial cell migration and proliferation. Bovine retinal capillary endothelial cells were selectively cultured from collagenase-digested microvessel fragments. In a filter system for the assessment of migration, endothelial cells responded to substrate-bound fibronectin but not to soluble fibronectin. Cell migration on collagen- or gelatin-coated filters was minimal, and these cells failed to adopt a spread morphology, remaining instead as round cells. Cell replication was quantified using a protein dye binding assay for adherent cells in 96 well plates. Serum was essential for growth irrespective of the substrate. Cells harvested from microvessel cultures proliferated more rapidly on collagen- and gelatin-coated plastic than on fibronectin and were unaffected by additions to the medium such as endothelial cell conditioned medium, whereas cells proliferating directly from the microvessels grew at a faster rate on fibronectin and also responded to conditioned medium supplement. When cultured on collagen gels, initial microvessel cells and harvested cells required surface fibronectin in order to adopt a cobblestone morphology. These results show that fibronectin is a requirement for bovine retinal capillary endothelial cell migration, but proliferation of these cells can be supported, with slight differences, by both fibronectin and collagen provided serum growth factors are present. These findings are relevant to the early phase of angiogenesis in which migration and proliferation of endothelial cells occurs.

Effects of soluble mediators generated during growth of the Landschütz ascites carcinoma on the chemotaxis of normal and Corynebacterium parvum-stimulated peritoneal leucocytes.

The influence of ascites fluid on the chemokinetic and chemotactic responses of mouse peritoneal leucocytes was investigated using modified Boyden chambers and the 'leading front' technique. The migration of normal cells towards endotoxin-activated mouse serum was significantly enhanced at high concentrations of fluid (40 and 20%) but was inhibited at lower concentrations (5 and 1%). By comparison, the chemotactic responsiveness of cells harvested 3 days after Corynebacterium parvum (C. parvum) injection was uniformly and more markedly increased over a wide range of fluid concentrations (0.1-50%). These effects were attributed to the presence of strong chemokinetic and chemotactic material in ascites fluid, which was not inactivated by heating. The activity was also demonstrated in the serum of tumour-bearing animals, was present to a much lesser extent in inflammatory exudate fluid and was absent from normal mouse serum. Detailed morphological studies of migrating cells revealed that although the ascites fluid contained a potent chemo-attractant for neutrophils, it inhibited the migration of mononuclear cells.

Retina contains endogenous heparinase activity.

Heparin binding growth factors (HBGF), a major component of retinal endothelial cell growth factor activity, have been located particularly to a storage site in the vascular basement membrane, where they are bound to heparan sulphate in a relatively inactive state. It has been suggested that angiogenic activity in the retina may be released, during basement membrane degradation, by heparinase enzymes. We now report that mitogenic saline extracts of retina contain heparinase-like activity, whereas non-mitogenic retinal homogenate does not. However, heparinase-like activity may only account for part of the mitogenic activity in retinal extract. In previous studies we have shown that the absence of mitogenic activity in retinal homogenate is due to a heat-labile inhibitor. Heat-treatment of retinal homogenate is due to a heat-labile inhibitor. Heat-treatment of retinal homogenate restored mitogenic activity to 60% of that in retinal extract, but this heat-treated homogenate still lacked heparinase activity.

Investigation of the procoagulant activity present in ascitic fluid and serum of mice bearing the Landschütz ascites carcinoma.

The direct procoagulant activity (PCA) of murine tumour cells was found to be more than three orders of magnitude greater than an equivalent concentration of either resident or Corynebacterium parvum-elicited, exudate peritoneal cells. Similarly, a soluble PCA was detected in the extracellular culture medium of only the tumour cells. Studies on the procoagulant nature of the serum and ascitic fluid of tumour-bearing animals suggested that the ascitic fluid may contain a unique PCA factor(s). This activity could not, however, be resolved from inherent procoagulant factors, either by gel filtration or ammonium sulphate fractionation, and a more specific assay for, say, a single enzymic reaction in the coagulation cascade would be required to identify the tumour-associated activity.

Serum biochemical changes in c. parvum-injected mice bearing the Landschütz ascites carcinoma.

Mice injected i.p. with 1.4 mg Corynebacterium parvum (C. parvum) displayed minor transient impairment of hepatic function, as evidenced within 5-8 days by significant elevations in serum aspartate aminotransferase and reductions in total protein, albumin and alkaline phosphatase. Inoculation of animals with the Landschütz ascites carcinoma (LAC) caused similar but more marked and prolonged liver injury with serological evidence of nephrotoxicity at advanced stages of tumour growth. Injection of C. parvum 24 h before tumour had no further influence on these serological changes observed in animals given tumour alone. No evidence was obtained to support histological findings that the LAC inhibits the granulomatous inflammatory response to C. parvum in mouse liver.

Influence of tumour carriage on the production of lymphokines affecting macrophage behaviour.

Normal and cyclophosphamide (Cy)-enhanced delayed-type hypersensitivity (DTH) reactions to sheep red blood cells (SRBC) were severely impaired in CD1 mice injected with 10(6) viable Landschütz ascites carcinoma (LAC) cells at the time of immunization. The tumour-induced suppression of DTH was accompanied by inhibition of splenic T-lymphocyte responses to antigen and mitogen. This was assessed by measuring lymphocyte transformation and the production of two lymphokines affecting macrophage behavior, i.e. lymphocyte-derived chemotactic factor (LDCF) and macrophage procoagulant-inducing factor (MPIF). Our results imply that impaired production/function of lymphokines affecting macrophage behaviour may play a key role in tumour-induced suppression of cell-mediated immunity and that this inhibition is independent of Cy-sensitive suppressor cells.

Tumour-induced suppression of delayed-type hypersensitivity in the mouse: independence of cyclophosphamide-sensitive suppressor cells.

Delayed-type hypersensitivity (DTH) responses to sheep red blood cells and ovalbumin were markedly reduced in mice receiving viable Landschütz ascites tumour cells at the time of immunization. Similar inhibitory effects were observed in control immunized animals when cell-free ascitic fluid or tumour bearer serum was administered together with antigen at the challenge site. Normal mouse serum exhibited much less pronounced inhibitory activity over the range of concentrations tested. High-dose cyclophosphamide (Cy) prior to immunization enhanced DTH responses to both antigens in normal mice. However, in animals also given tumour or soluble tumour-associated material, the immunosuppressive effect which had been observed in non-Cy-treated controls was preserved. It is concluded that the observed suppression of DTH is not dependent on Cy-sensitive T suppressor cells or on the presence of immunosuppressive factors during induction of the immune response.

Inhibition by the Landschütz ascites carcinoma of the granulomatous inflammatory response to C. parvum.

I.p. or i.v. administration of Corynebacterium parvum (CP) to MF1 mice induces a generalized inflammatory response, associated with marked hepatosplenomegaly and accompanied by a pronounced granulomatous response in the liver. Injection of the Landschütz ascites carcinoma (LAC) 24 h after CP substantially reduced the intensity of the inflammatory response, and decreased both the frequency and size of the hepatic granulomas, as revealed by morphometric analysis of histological sections. The difference in cellular composition of the granulomas between the experimental groups, as revealed by light microscopy, was further emphasized and characterized by ultrastructural studies. These revealed the predominance of macrophages within the granulomas in tumour-bearing mice, in contrast to the predominance of epithelioid cells in the lesions which developed in mice given CP alone. Our experimental findings show that the inhibitory effect of the growing LAC on granuloma formation in response to CP cannot be ascribed to (a) sequestration of the microorganism within the growing tumour, (b) a nonspecific inflammatory stimulus, (c) diversion and seqestration of mononuclear phagocytes in the growing tumour or (d) the presence of lactate dehydrogenase-elevating virus in either the host or tumour cells. The inhibition of liver granuloma formation is consistent with an effect mediated by soluble, heat-stable tumour-associated factor(s).

Effects of the Landschütz ascites carcinoma and ascitic fluid on macrophage activity in C. parvum-injected mice.

I.p. administration of 1.4 mg Corynebacterium parvum (C. parvum) 24 h before inoculation of Landschütz ascites carcinoma (LAC) cells significantly impaired growth of the tumour in MF1 mice. The injection of tumour cells caused a transient inhibition of the activity of the mononuclear phagocyte system (MPS) in both normal and C. parvum-treated hosts, as evidenced by impaired clearance of colloidal carbon from the bloodstream and reduction in hepatic phagocytosis of 51Cr-labelled sheep erythrocytes. Depression in Kupffer-cell activity was associated with a shift in particle distribution towards the spleen. The pronounced hepatosplenomegaly in response to C. parvum was significantly less in animals which also received tumour cells. Histological examination of liver and spleen revealed evidence of depressed MPS activity. Granuloma production in the liver in response to C. parvum was inhibited in tumour-bearing mice, and macrophage proliferation within the spleen was also reduced. Ascitic fluid showed similar inhibitory effects to those of tumour-cell suspensions, suggesting production by LAC of a heat-stable macrophage-inhibitory factor.

Tumours acquire their vasculature by vessel incorporation, not vessel ingrowth.

This study was designed to investigate the early development of tumour circulation using a transplantable mouse mammary adenocarcinoma. Tumour cells (10(6] were injected subcutaneously into the flank and groups of treated mice were killed at 24 h intervals up to 12 days; the tumours and surrounding tissues being processed by standard histological methods. Sections of tumour were sub-divided into neoplastic tissue, vessels and connective tissue using computer-assisted morphometric techniques: the host tissue around the tumour was similarly quantified for vessels and connective tissue. With increasing tumour size, the proportion of vessels within tumours rapidly increased to reach a plateau of approximately 1.5 per cent of tumour volume, a 400 per cent increase on the vascular density of normal subcutaneous tissue. Within tumours, vascular density was always higher at the periphery than the centre. The most pronounced increase in vascular density affected the host tissue around the tumour. It is not clear why vascular development is most prominent outwith the tumour when postulated angiogenic factors, such as tumour angiogenesis factor, are presumably released within. Our results imply, however, that tumours acquire their vasculature by infiltration into, and expansion between, a network of newly formed vessels in the surrounding connective tissue.