Robert M. Fauve

Maintenance of granuloma macrophages in serum-free medium.

Granulomas were induced by injecting polyacrylamide beads into subcutaneous pouches created by divulsion of the dorsal skin of mice. More than 10(7) phagocytic cells (60% macrophages, 40% polymorphonuclear cells) could be recovered from this granuloma. The separation of the phagocytic cells can be achieved either following sedimentation in Percoll or following the incubation of cells on plastic petri dishes. Phagocytosis of zymosan was observed in macrophages maintained in vitro for 1 month in Eagles serum-free medium.

Macrophage Spreading: Inhibition in Delayed Hypersensitivity

The capacity of peritoneal macrophages to spread was studied with cells of mice infected with Listeria monocytogenes and with cells of guinea pigs sensitized with BCG (bacille Calmette Gu�rin) vaccine or immunized with ovalbumin. In macrophages taken from animals having delayed hypersensitivity, this ability was markedly decreased by the presence of specific antigen for less than 1 hour. Such an effect was not observed in guinea pigs having only circulating antibodies.

Remote effects of inflammation on non-specific immunity

Following inflammation induced in mice with non-biodegradable, non-diffusible, and non-antigenic substances, host resistance is increased against bacteria, parasites and malignant cells injected at a distance from the inflammatory focus. This resistance is also increased in germ-free and nude mice. The increased resistance is correlated with (1) an increased leukopoiesis induced, at least in part, by a protein (MW = 40 kDa, pI = 5.2) which, in vitro, is able to induce the differentiation of bone-marrow cells into polymorphs; (2) the occurrence of giant cells in the granuloma which, after incubation in vitro, release an immunostimulating protein able to activate mice macrophages in vivo; (3) activation in vivo of liver and spleen macrophages following the occurrence, both in granuloma and in serum, of a protein (MW = 56 kDa, pI = 5) which, contrary to endotoxin, is heat-labile and can fully protect mice against Listeria monocytogenes. Furthermore, this protein, which is different from TNF and GM CSF, is able to activate macrophages against Lewis tumor cells. The same protein can be isolated from rat and is antigenically related to a human protein having the same biological activity.

Inflammation and host resistance against pathogens.

After a review of all factors released in an inflammatory focus which act on pathogens, a summary of recent findings concerning the influence of an inflammation on the remote control of mouse resistance against pathogens is given. In treated mice, the ability of spleen and liver macrophages to killListeria was found to be increased between 3 and 18 days following the induction of the inflammation. An increased resistance against the Lewis carcinoma was found, even when the mice were treated one day after the inoculation of malignant cells. In the treated mice, an increased cellular multiplication was observed in bone marrow, thymus, spleen, lymph nodes whereas no variations were observed in other tissues. The higher host resistance is correlated with an increased leucopoiesis and the activation of liver and spleen macrophages. The serum of treated animals is ablein vitro to activate peritoneal macrophages against Lewis carcinoma cells, to stimulate the multiplication of bone marrow cells and to agglutinate malignant cells. In order to explain the mechanisms of such an increased host resistance, investigations were undertaken to isolate and characterize possible active substances in the granuloma and in the serum from granuloma-bearing mice, and to check the possible activity of mediators of inflammatory reactions. Concerning the granuloma and the serum, it was possible, following electrofocusing, to extract and to isolate a fraction which is able to protect mice against a lethal inoculum ofListeria. Among the known mediators of acute inflammation, it was found not only that kinins are able to increase the spreading of macrophagesin vitro but that kinins or some of their degradation products are able,in vivo, to increase the resistance of mice againstListeria and the Lewis carcinoma. These results emphasize the importance of local non-specific inflammatory reactions on macrophages.

Influence of Synthetic Polynucleotides on the Bactericidal Power of Mouse Macrophages Against Listeria Monocytogenes

In 1964, Braun and Kessel (1964) suggested that bacterial endotoxins may release stimulatory oligonucleotides from intracellular environments, the release mechanism being the capacity of endotoxins to alter or to damage mammalian cell membranes and cells. More recently, Braun and Nakano (1967) were able to support this assumption, and demonstrated that complexes of polyadenylic and polyuridylic acids or polycytidylic and poly-guanylic acids enhanced the early rate of increase in numbers of antibody-forming spleen cells in mice immunized with sheep red blood cells or other particulate antigens. The same year, Lampson et al. (1967) reported that double-stranded ribonucleic acids derived from several sources were active inducers of interferon and of enhanced host resistance to viral infection. Since then, it has been shown that synthetic double-stranded polynucleotides, such as poly I:C and poly A:U, can increase the resistance of the host against several viral infections and experimental tumors as will be reported in this Symposium.

The anti‐tumoral activity of human glycoprotein HGP.92: A study with the mouse Lewis‐lung‐tumor cell

The ability of a purified human glycoprotein (HGP.92) to exert anti‐tumor activity was investigated in a mouse model using long‐term readout assays. In vitro, in the presence of inflammatory mouse macrophages incubated with HGP.92, the growth of the mouse Lewis‐lung‐tumor cells (3LL) was decreased. This effect was concentration‐dependent and required direct contact between tumor targets and HGP.92‐treated macrophages. In addition, if the macrophage monolayer was depleted of HGP.92 before addition of the target cells, no more cytostatic effect was observed. This anti‐tumor activity of HGP.92‐treated mouse macrophage was partially abrogated by addition of catalase in the culture medium, but not by superoxide dismutase or scavengers of the hydroxyl radical and singlet oxygen. Moreover, this tumor‐cell growth reduction was not dependent on nitric oxide. In vivo, multiple i.v. injections of HGP.92 (5 times, 3 days apart) during the first week and a half exerted significant anti‐tumor activity, as assessed by the reduction of both the number and the size of the lung nodules 3 weeks after i.v. inoculation of 3LL cells. Int. J. Cancer, 70:0–0, 1997.

Effects of Nonspecific Inflammation on the Reticuloendothelial System

The first review concerning the influence of inflammation on host resistance was written by Metchnikoff (1892). He had already pointed out the significance and the importance of the mobilization of amoebocytes in primitive metazoans facing an aggression, and later on, showed that phagocytosis was a cornerstone of host resistance. As reviewed again by Metchnikoff (1901) in Immunity in Infectious Diseases, he made it clear that inflammation is one of the oldest phylogenetic mechanisms of resistance. In the same book, it was also reported that an increased resistance against bacteria can be induced at the site of an inflammatory reaction. Indeed, it had been observed during the last two decades of the 19th century that animals injected in the peritoneal cavity with different nonantigenic phlogogens are able to survive a lethal inoculum of pathogens. The explanation given for such an increased resistance was the increased influx of phagocytic cells into the site of infection. He also regarded these inflammatory phagocytes as being stimulated. Today, it is known that these phagocytic cells are not the only effectors responsible for such stimulation; nevertheless, the importance of inflammation as a nonspecific resistance mechanism is now well established. Moreover, it is also widely accepted by immunologists interested in in vivo immunity that specific immune effectors (antibodies and lymphocytes) are responsible for the induction of inflammatory reactions in the vicinity of specific targets. Since macrophages are among the most potent actors in host-invader relationships, one may wonder what elements of the tangled web of inflammation can modulate macrophage functions. In order to answer this question, we will consider, in the inflammatory focus, the different modulators of macrophages.

Non-specific Inflammation and Host Resistance Against Pathogens

Following an inflammation induced in mice with non biodegradable, non diffusible and non antigenic substances, the host resistance is increased against bacteria, parasites and malignant cells injected at distance from the inflammatory focus. This resistance, which is also increased in germ-free and nude mice, is correlated with an increased leukopoiesis, the activation of macrophages and the presence in the granuloma and in the serum of substances able, in vivo and in vitro, to activate macrophages, to agglutinate malignant cells and to stimulate the proliferation of bone marrow cells. Furthermore, kinins and some of their split products are able to stimulate the host resistance.

Inflammation and host resistance to pathogens.

Among the early events occurring after the entry of pathogens into the tissues, inflammation is one of the mechanisms which helps the host to increase the number and the quality of the weapons against aggressors. The purpose of this short review is to summarize briefly the different stages of an inflammatory reaction and to describe some of the mechanisms by which pathogens escape increased host resistance. Furthermore, it will be shown that a local inflammation, distant from the site of aggression can markedly increase the resistance of the host to pathogens such as bacteria, parasites and tumour cells.

11 – AN IN VITRO TEST FOR DELAYED HYPERSENSITIVITY: MACROPHAGE SPREADING INHIBITION (M. S. I.)

Inhibition of macrophage spreading in vitro (M. S. I.) occurs if peritoneal cells from mice or guinea pigs which exhibit delayed hypersensitivity are incubated in the presence of the specific antigen. Such inhibition has been observed with Listeria antigens ( 1 ), tuberculin and some streptococcal antigens ( 2 ). Recently, inhibition was shown also with mice immunized with Trypanosoma cruzi ( 3 ). This test can perhaps be used also with rats and it may be of some help in detecting transplantation antigens. The test is limited to an incubation time of 30 minutes. Longer times of incubation result in macrophage clumping. Typical results are given in reference ( 2 ). Several sensitive and normal animals must be tested simultaneously.