Donatella Taramelli

Measurement of macrophage-mediated cytotoxicity against adherent and non-adherent target cells by release of 111indium-oxine

This report describes the utilization of 111 indium-oxine chelate ([111In]Ox) for studies of macrophage-mediated cytotoxicity. [111In]Ox efficiently labeled both non-adherent and adherent tumor targets with no decrease in cell viability. Spontaneous release of intracellularly incorporated [111In]Ox was very slow (0.25-0.50%/h) from most targets, making isotope-release assays of at least 48 h feasible. In addition, released [111In]Ox was not reutilized. In contrast to its low spontaneous release from intact cells, incorporated [111In]Ox was rapidly released from tumor targets after interaction with activated macrophages. Levels of [111In]Ox released in response to cytolytic macrophages correlated well with those observed for the 51Cr and [3H]TdR radiolabels. Therefore, [111In]Ox can be utilized for relatively short-term (less than 20h) assays with lymphoma targets, as well as for longer-term assays with adherent cells. This should facilitate the testing, with the same radioisotope-release assay, of a wide range of tumor targets for susceptibility to macrophage-mediated cytotoxicity.

Induction of immunotoxicity by polycyclic hydrocarbons: Role of the Ah locus

We have employed the plaque forming cell (PFC) response to sheep erythrocytes as well as lymphocyte proliferation to study the induction of immunotoxicity in AHH-inducible (Ah Locus positive, C57BL/6N; B6C3F1) and AHH non-inducible (Ah Locus negative, DBA2/N) mice following administration of polycyclic aromatic hydrocarbons. When two potent carcinogenic polycyclic hydrocarbons which induce AHH activity, 3-methylcholanthrene (MCA) or 1,2,5,6-dibenzanthracene [DB(a,h)A] were administered IP, immunotoxicity was observed in both AHH-inducible and AHH non-inducible animals. However, the AHH-inducible animals appeared to be more sensitive, and substantial suppression of a PFC response toxicity could be induced with doses as low as 14 mg/kg methylcholanthrene. While suppression of a mitogen response required a dose of 43–125 mg/kg. Administration of the weak carcinogen 1,2,3,4-dibenzanthracene [DB(a,c)A], IP, which similarly induces AHH activity in inducible animals, failed to induce immunotoxicity in either C57B1/6N or DBA/2N mice. In contrast to the results obtained following IP administration, when MCA was administered repeatedly (4X) via an intragastric (IG) route we observed striking immunosuppression of a PFC response in Ah locus negative (DBA/2) animals but minimal effects in Ah locus positive animals (C57B1/6). We finally observed that a single IP dose of MCA (125 mg/kg) to Ah locus positive animals substantially inhibited Natural Killer Cell activity but had more limited effects on the ability of an animal to reject a challenge by an immunogenic syngeneic fibrosarcoma.

Endotoxin requirement for macrophage activation by lymphokines in a rapid microcytotoxicity assay

A short term microcytotoxicity assay is described for studying the activation of M0 by lymphokines. Proteose-peptone-induced macrophages were purified by adherence in flat-bottomed microtiter plates and then activated by MAF-containing supernatants for 18 h. 51Cr-labeled tumor target cells were added for an additional 18 h, and then the supernatants were harvested and the % isotope release quantitated. When endotoxin-free medium and FBS were used, we found that small amounts of LPS were absolutely required for macrophage activation in this assay. The advantages of this technique included (a) good reproducibility, (b) the requirement for small number of M0, and (c) the potential of standardizing the assay and thereby testing a large number of samples. Moreover, this assay may have particular value for investigations of M0 activation by exogenous stimuli, since M0 that were not pretreated with activating agents did not exhibit cytotoxicity.

Suppression of lymphokine production: II. Macrophage-dependent inhibition of production of macrophage activating factor.

Abstract The ability of different populations of macrophages to affect the production of macrophage activating factor (MAF) by stimulated T lymphocytes was investigated. We found that activated macrophages, infiltrating MSV-induced regressing tumors or macrophages recovered from the peritoneum of mice injected with Corynebacterium parvum, were able to actively suppress the production of MAF. MAF production by antigen-stimulated MSV-immune or -alloimmune spleen cells and by normal spleen cells stimulated by Con A was susceptible to macrophage-dependent suppression to a similar extent. In contrast, resident macrophages or those elicited by light mineral oil or proteose-peptone did not affect MAF production. While suppressor macrophages added at the time of the lymphocyte stimulation inhibited MAF production, the same cells added 4–6 hr after stimulation were ineffective. Therefore, it seems that the macrophages suppressed the early events of lymphocyte activation leading to MAF production. Suppressor macrophages, by inhibiting MAF production, may limit the expansion of the cytotoxic activity. This regulation of macrophage functions, mediated by the effects of suppressor macrophages on T lymphocytes, could be responsible for an insufficient antitumor cytotoxic response by macrophages.

Lymphokines inhibit macrophage RNA synthesis

The effects of lymphokine (LK) preparations on the incorporation of [3H]uridine into macrophage RNA were investigated. Supernatants from murine spleen cells activated in vitro by alloantigens or Con A, and shown to contain macrophage-activating factor (MAF), were used as the source of LK. It was observed that such LK preparations contain factor(s) causing a profound inhibition of [3H]uridine incorporation into the RNA of proteose-peptone-elicited peritoneal macrophages. Such RNA-labeling inhibitory factor (RIF) was absent in control supernatants from nonstimulated cultures, and showed activation curves similar to that of MAF. RIF activity was not due to altered permeability of macrophages to [3H]uridine nor to the changes in the specific activity of the pool of RNA precursors, but rather reflected an altered metabolism of RNA. The inhibition of RNA synthesis was dependent upon the presence of nanogram amounts of LPS as a costimulator. Moreover, the response to RIF appeared to be genetically controlled since macrophages from C3H/HeJ mice were not affected by RIF, while C3H/HeN mice were fully responsive. In parallel cultures of macrophages, LK were also tested for their MAF activity, and a strong similarity between the biological conditions in which MAF and RIF activities were expressed could be demonstrated. The assay for RIF provides a new and convenient parameter for measuring macrophage-sensitive LK activity that might be very useful for monitoring purification or for screening of T-cell hybridoma supernatants.

SUPPRESSION OF MURINE NATURAL KILLER CELL ACTIVITY BY NORMAL PERITONEAL MACROPHAGES

Publisher Summary This chapter discusses suppression of murine natural killer (NK) cell activity by normal peritoneal macrophages. It has been suggested that natural killer (NK) cells have a wide range of biological activities, including immunosurveillance against neoplastic cells, protection against viral infections, and production of lymphokines such as interferon (IFN). In an experiment discussed in the chapter, the possibility that normal macrophages regulate NK activity was examined, as NK activity is present in normal animals. To examine the ability of various Mφ populations to suppress NK activity, Mφ from the peritoneal cavity and spleen of both normal and Corynebacterium Parvum (C. parvum)-injected mice were compared. The results demonstrate that normal peritoneal Mφ have the ability to suppress either spontaneously maintained or IFN-augmented NK activity in vitro. Although the potential in vivo regulatory role of Mφ on NK activity has not been established, it is tempting to speculate that peritoneal Mφ may control the level of NK cell-mediated cytotoxicity. Normal peritoneal Mφ has the ability to suppress NK activity in vitro and this ability is altered by injection of C. parvum.

Activation of Tumoricidal and/or Suppressor Macrophages: Different Stimulatory Signals Trigger Either Function both in Vivo and In Vitro

Macrophages upon in vivo and in vitro stimulation become activated and manifest functional and morphological characteristics either not present or differently expressed in non-stimulated macrophage populations (1–4). Antithetical functions can be exerted by a given macrophage population. For instance, cytotoxic macrophages could contribute to the elimination of infectious agents or to the control of neoplastic growth. On the other hand, by inhibiting certain immune responses, suppressor macrophages could impair host defense mechanisms.