Marcella Lipari

Evidence for three binding sites for C3 (hemolytically inactive), C3b and C3d on a CR2-positive Burkitt lymphoma-derived cell line (Raji)

The present investigation shows that C3 (hemolytic inactive) as well as C3b and C3d bind Raji, a CR2‐positive Burkitt lymphoma‐derived cell line. Pretreatment of the cells with OKB‐7 inhibited the binding of C3, whereas pretreatment with HB‐5 inhibited the binding of C3b. Furthermore, the cells coated either with OKB‐7 or HB‐5 bound high amounts of C3d. TPA‐treated cells showed binding for C3b and weak binding for C3 and C3d. Taken together, the data suggest that Raji cells may express three binding sites for C3, C3b and C3d which can be differently modulated by anti‐CR2 MoAbs and TPA.

Stimulation of macrophages with IFNγ or TNFα shuts off the suppressive effect played by PGE2

PGE2 has been shown to be able to interfere with various lymphocyte and macrophage functions, but its effects on macrophage activation are still unclear. In this study, carried out on peritoneal macrophages obtained from healthy, tumour-bearing and Corynebacterium parvum-treated mice, we demonstrated that PGE2 is involved in the down-regulation of macrophage activation, but it cannot exert its inhibiting effect when macrophages are further stimulated with activating cytokines, such as IFNγ and TNFα. Our findings provide new insight into how macrophage tumoricidal activity may be induced and maintained even in presence of significant levels of PGE2.

Lewis Lung Carcinoma Cells Enhance the Synthesis of C3 and are Opsonized by C3 Secreted from Murine Macrophages

We investigated the effect of Lewis Lung carcinoma cells on the production of C3 by murine macrophages and examined the capacity of secreted C3 to opsonize Lewis Lung carcinoma cells. C3 released in culture from macrophages obtained from tumor-bearing C57Bl/6 mice as well as from normal macrophages exposed to Lewis Lung carcinoma cells in vitro was measured by hemolytic assays and by Western blot. We found that contact with tumor cells in vivo as well as in vitro enhanced the amount of C3 secreted by murine macrophages by a factor of 2-3. The inflammatory agent carrageenan caused only a small increase in the amount of secreted C3. On Western blots of concentrated macrophage supernatants, there was partial cleavage of secreted C3 which was, however, not more pronounced in the case of C3 from tumor-stimulated macrophages than from normal macrophages. Supernatants from normal as well as tumor-stimulated macrophages were capable of opsonizing Lewis Lung carcinoma cells as shown by their capacity to bind human erythrocyte in an immune adherence reaction. Pretreatment of the tumor cells with a protease inhibitor, PMSF, inhibited the capacity of the tumor cells to bind C3, suggesting that a tumor cell-associated protease might be involved in the binding of C3 to the tumor cell surface.

C3 Synthesis and CRs Expression during DiHerentiation of a Murine Stem Cell Line

C3 production, release and CRs expression during the neutrophilic differentiation of a murine non tumorigenic cell line is investigated. The murine non tumorigenic cell line 32DCl3(G) which undergoes terminal differentiation into polymorphonuclear granulocytes when cultured in presence of G-CSF was selected as a suitable in vitro model for this study. The results show that as the cells progress into the differentiation program, levels of C3 mRNA increase, accompanied by increased C3 production. As differentiation progresses the cells gradually express CRs on their surface; these are undetectable on the surface of undifferentiated cells. As a consequence of CRs appearance, cells become able to bind C3 through receptorial binding. Differences were found in the modality of C3 secretion: differentiated cells tend to store C3 in their intracellular compartments rather than secrete it continuously into the medium and they respond to membrane stimulation with increased secretion of C3. Treatment of 32DCl3(G) with TNF-alpha increased C3 production in a time- and dose-dependent fashion. Cell response to this stimulus progressively increases during the differentiation process suggesting that they acquire functionality in the signal transduction mechanisms.

IFNγ and TNFα cause an increased release of C3 by murine macrophages

Abstract Macrophages from mice bearing Lewis lung carcinoma release higher amounts of C3 molecules than macrophages from healthy mice. The C3 pro-releasing activity operating in vivo was suspected to be due to an immunological network. Indeed, the supernatants of splenocytes from tumor bearing mice, but not from normal mice, induced in vitro an increased release of C3 molecules by macrophages. Recombinant IFNγ and TNFα were strong inducers of C3 release, while IL-2 acted poorly. The C3 pro-releasing activity of splenocyte supernatants was largely prevented by their pretreatment with specific mAb anti IFNγ or anti TNFα, but not completely prevented by the simultaneous neutralization of the two cytokines. Taken together, these results show that murine macrophages increase the release of C3 molecules upon treatment with IFNγ or TNFα and that these cytokines released in vivo by splenocytes from tumor bearing mice may account, together with a yet unknown factor, for a humoral network causing the increased release of C3 by peritoneal macrophages.

Chemotactic response of rat macrophages is enhanced by two diastereoisomers of LTB4

We demonstrated that rat peritoneal cells synthesize, and release into the culture medium, substances that elicit a chemotactic responsiveness of rat macrophages. These substances were identified by HPLC analysis as two diastereoisomers of LTB4, precisely delta-6-trans-LTB4 and delta-6-epi-trans-LTB4. Peritoneal cells release, also, other lipoxygenase metabolites such as LTD4 and LTB4, which were shown not to be active in eliciting chemotaxis of rat macrophages, in agreement with data of other authors. Quantitative assays for the measurement of PMNs or macrophage chemotaxis were carried out in Boyden chambers. Release of leukotrienes by rat peritoneal cells into the culture medium was strongly enhanced upon stimulation with calcium ionophore A23187, an agent known to increase cytoplasmic Ca2+ concentration, thus leading to the activation of Ca2+-dependent phospholipase and a consequent increase in the amount of endogenous free arachidonic acid available for biotransformation. The action of several inhibitors of arachidonate metabolism at concentrations more selective for the lipoxygenase than the cycloxygenase pathway, was also studied. alpha-Tocopherol and ASA were shown to be the most selective in inhibiting the synthesis of both the LTB4 diasteroisomers active as enhancers of rat macrophage chemotaxis.

C3-induced 3LL cell proliferation is mediated by C kinase

It has been demonstrated that the third component of complement (C3)1 and its peptides increase normal and tumour cell proliferation. However, the signal cascade responsible for this phenomenon is still unknown. In this study, we elucidate some of the mechanisms involved in the signalling of C3 stimulation of cell proliferation. We have first investigated the in and out traffic of C3 peptides, then we have identified the subcellular localisation of internalised C3 and, finally, we have explored the role of protein phosphorylation in C3 traffic and in the proliferation of the Lewis lung carcinoma (3LL) cells. Our results indicate that traffic of C3 is not dependent on cytoskeletal integrity and requires protein kinase C‐dependent phosphorylation. In addition, proliferation of 3LL cells stimulated by C3 depends on both C3 internalisation and protein‐kinase C phosphorylation.

Macrophage Tumor Cell Interaction is Enhanced by C3 Fragments

We tested the capacity of Lewis Lung carcinoma cells (3LL) to activate the alternative pathway of complement and to bind the C3 fragments on the plasma membrane. C3 fragments were detected by cytofluorometry and by immunoblotting. In time, the fixed C3b molecules were further cleaved into iC3b. The presence of C3b/iC3b on the target enhanced the formation of conjugates with macrophages. In spite of increased contacts, macrophages from tumor bearing mice were not cytotoxic. Only preactivated macrophages, by in vivo treatment with Corynebacterium parvum, were shown to be cytotoxic; this function was potentiated when the target cells were opsonized with C3b/iC3b.

Role of PGE2 Produced by Neoplastic Cells as Modulators of Macrophage Chemotaxis

The relationships between tumor cells and macrophages during tumor growth have been the subject of many investigations. The aims were essentially the following: a) a better understanding of the mechanism(s) leading to the accumulation of macrophages within a growing tumor and b) the understanding of the mechanism(s) responsible for the macrophages’ acquisition of peculiar properties (strictly resembling those which occur during the process of activation) in tumor-bearing subjects.

A comparative analysis of macrophage activation in C57Bl/6 mice treated with inflammatory compounds or bearing Lewis lung carcinoma

SummaryThe different percentages of acid hydrolases released by peritoneal macrophages obtained from C57Bl/6 mice injected i.m. with talc, carrageenan or 3LL cells, and also the demonstration that hydrolase secretion fromin vitro macrophage monolayers of tumor-bearing mice undergoes a further increase when zymosan is added to the culture, strongly suggest the possibility that more than one membrane surface receptor is involved in the triggering of lysosomal enzyme release. Furthermore, there is evidence from the reported results that, besides immune complexes, neoplastic cells or their metabolic product(s) could act as inducers of the lysosomal enzyme secretion by macrophages.