Michel Denis

Human monocytes/macrophages: NO or no NO?

The production of nitric oxide (NO) and other reactive nitrogen intermediates by cytokine‐activated rodent cells is an important component of antimicrobial and/or antineoplasic activity of these cells. This pathway involves the oxidation of arginine to citrulline, with the concomitant release of NO by an inducible form of NO synthase (iNOS). Numerous cell types express iNOS after stimulation with bacterial products and/or cytokines. The role of NO and its derivatives in host resistance is a subject of intense investigation in mouse models of infections or neoplasia. Although human cells such as hepatocytes and endothelial cells have been shown to express an inducible NO synthase, the presence of such a pathway in human monocytes/macrophages has been questioned by many investigators and is a subject of great controversy. In this short review, we discuss some salient points of this debate. J. Leukoc. Biol. 55: 682–684; 1994.

Human neutrophils activated by interferon-gamma and tumour necrosis factor-alpha kill Entamoeba histolytica trophozoites in vitro.

The interaction between cytokine‐activated human neutrophils and Entamoeba histolytica trophozoites was studied as well as the mechanism(s) involved. Treatment of neutrophils with rlFN‐γ alone allowed them to kill 30% of E. histolytica trophozoites; however, rlFN‐γ and rTNF‐α pretreatments in combination increased neutrophil killing to 70%. In the absence of direct contact between neutrophils and amebae, rlFN‐γ‐treated neutrophils were shown to kill 70% of amebae, and rlFN‐γ‐ and rTNF‐α‐treated neutrophils killed 97% of amebae. Neutrophil enhancement of amebicidal activity following cytokine treatments was correlated with Increased neutrophil resistance to amebic contact‐dependent killing and was shown to be 73% H2O2 dependent.

THE INVOLVEMENT OF NITRIC OXIDE IN A MOUSE MODEL OF ADULT RESPIRATORY DISTRESS SYNDROME

The release of free radicals and pro-inflammatory cytokines such as nitric oxide (NO) and tumor necrosis factor alpha (TNFα) is commonly observed in adult respiratory distress syndrome (ARDS) following infection or exposure to microbial products. The aim of this study was to scrutinize the involvement of NO in ARDS in a mouse model determined by the sequential exposure to lipopolysaccharide (LPS) and formyl-norleucyl-phenylalanine (FNLP). Nitrite measurements in bronchoalveolar lavage fluids (BALF) and sera demonstrated that exposure to microbial products elicits large amounts of NO in LPS/FNLP-challenged mice. This release was significantly inhibited by infusion with the inducible NO synthase antagonist, aminoguanidine (AG). Our results show that LPS/FNLP exposure induces lung damage as demonstrated by protein and lactate dehydrogenase (LDH) increases in BALF. Liver damage was also detected in LPS/FNLP-challenged mice with increases in serum ornithine-carbamoyltranferase (OCT) levels. LPS/FNLP infusion led to elevated levels of the cytokines interleukin-6 (IL-6) and tumor necrosis factor alpha (TNFα) in the sera. LPS/FNLP also led to neutrophil adhesion in the lung vasculature, as seen by increased levels of myeloperoxydase. Interestingly, inhibition of NO release in challenged mice led to an important increase in markers of tissue damage in the lungs and livers, but a decrease in neutrophil recruitment. Infusion of AG in LPS/FNLP-challenged mice led to a much increased level of sera TNFα. These data suggest that after exposure to microbial products, NO generated as a result of activation of the inducible NO synthase blocks the full expression of tissue damage in the lungs.

Killing of Mycobacterium smegmatis by macrophages from genetically susceptible and resistant mice.

The bactericidal function of macrophages was investigated in congenic mice expressing the phenotype of susceptibility (B10.A, Bcgs) or resistance (B10.A.Bcgr) to mycobacterial infection. When splenic and peritoneal macrophages from these two mouse strains were infected in vitro with Mycobacterium smegmatis, the Bcgr macrophages were shown to inactivate M. smegmatis more efficiently than their Bcgs congenic counterparts. The mechanisms of this superior antimycobacterial activity was studied further. Addition of catalase did not abolish killing to a significant degree in either allelic type of macrophage, suggesting that hydrogen peroxide production was not involved in the killing activity controlled by the Bcg gene. Activation of Beg macrophages by exposure to crude lymphokines rendered them equally as efficient as their Bcgr counterparts in their capacity to destroy M. smegmatis. This finding suggests that both the genetically resistant and susceptible macrophages have the potential to kill M. smegmatis in vitro. This potential is expressed constitutively by the Bcgr but not Bcga macrophages and can be induced, by lymphokine treatment, in the Bcga macrophages. In a final set of experiments, the macrophage killing of M. smegmatis was evaluated as a test system to type for the Bcg gene allelic type in vitro, using a set of AXB and BXA recombinant inbred strains of mice. Results obtained show that typing of AXB/BXA recombinant inbred strains for the trait of bactericidal activity vs. M. smegmatis in vitro revealed a perfect match with the strain distribution pattern of resistance/susceptibility to Mycobacterium bovis BCG in vivo.

Antioxidant therapy partially blocks immune-induced lung fibrosis

A mouse model of hypersensitivity pneumonitis was generated by challenge with a thermophilic actinomycete. Oxygen radical scavengers were administered to challenged mice: vitamin E at 1000 units daily, polyethylene glycol-superoxide dismutase (SOD) at 500 units daily, polyethylene glycol-catalase at 10,000 units daily, 1,3,dimethyl-2-thiourea (DMTU) at 2 mg daily, and the biomimetic SOD, copper(II) [diisopropyl salicylate]2 (CuDIPS) at 1 mg daily. At three weeks after actinomycete challenge, a 10-fold increase in bronchoalveolar (BAL) cell number was observed. Treatments with catalase or DMTU were without effect on the BAL cell number in challenged mice. However, infusion of vitamin E was associated with an increased BAL cell influx (15-fold increase at two and three weeks). Similarly, treatment with PEG-SOD and CuDIPS resulted in an increase in cell number at two and three weeks. PEG-SOD or CuDIPS treatment resulted in a strong neutrophilia, whereas control challenged mice had a cellular influx mostly of macrophages and lymphocytes. Vitamin E treatment of challenged mice led to an increased T lymphocyte recruitment at two and three weeks. In vitro studies showed that actinomycete challenge was associated with an enhancement of alveolar macrophage O2− release, which was blocked by PEG-SOD, vitamin E, or DSC treatment but was unaffected by catalase or DMTU treatment. In control challenged mice, there was a 25-fold increase in the BAL albumin concentration at two weeks. PEG-SOD, vitamin E, or CuDIPS treatment all decreased the albumin concentration; the three modulators also diminished lung fibrosis at two or three weeks, as seen by a decrease in lung hydroxyproline and collagen synthesis by lung fibroblasts. Examination of sections from lungs of challenged animals showed evidence of cellular infiltrates around the bronchi and the blood vessels. Challenged mice given continuous infusions of vitamin E, SOD, or CuDIPS had lung histological scores that were significantly lower than control challenged mice or challenged mice treated with catalase or DMTU. Thus, therapies based on O2− scavenging or treatment with a general antioxidant such as vitamin E may hold some promise in the treatment of hypersensitivity pneumonitis.

In vivo activation of macrophages by IFN‐γ to kill Entamoeba histolytica trophozoites in vitro

Summary To determine the role of interferon‐gamma (IFN‐γ) in the activation of macrophages to kill Entamoeba histolytica trophozoites in vitro, C57BL/6 mice were injected with various doses of recombinant IFN‐γ (rIFN‐γ) by either the intravenous, intraperitoneal or intramuscular routes. Mice were treated with doses of rIFN‐γ ranging from 101 to 105 units. Twenty hours later, peritoneal macrophages were harvested from the treated animals. Macrophage monolayers were prepared and their in vitro cytotoxic activity against a virulent strain of E. hystolytica (IP:0682:1) was determined. Amoebicidal activity was determined by counting the number of dead trophozoites by Trypan Blue exclusion in cultures containing macrophages and amoebic trophozoites which were incubated together for 4 h. Both intravenous and intraperitoneal treatment resulted in the recovery of macrophages from the peritoneal cavity which exhibited amoebicidal activity in vitro. Peritoneal macrophages harvested from mice that had been treated intraperitoneally or intravenously with rIFN‐γ, however, showed significantly more amoebicidal activity in comparison to macrophages harvested from animals treated intramuscularly. There was a dose dependent relationship between the concentration of rIFN‐γ used to activate macrophages in vivo and the number of dead trophozoites in vitro. In addition, these results confirm our previous observations that treatment in vitro with rIFN‐γ can activate murine peritoneal macrophages to kill amoebic trophozoites.

Murine hypersensitivity pneumonitis: interleukin-4 administration partially abrogates the disease process

C57BL-6 inbred mice were given intranasal instillations of Faeni rectivirgula (150 micrograms/day, 3 days a week for 3 weeks) to produce a lung inflammatory reaction which mimics Farmers lung in humans. Challenged mice developed a strong inflammatory response in their lungs, based on various markers (lung index, bronchoalveolar cell number, fibrosis). The effect of interleukin-4 (IL-4) was studied by infusing mice intraperitoneally with 100, 1000 or 10,000 units of IL-4 weekly during the challenge period. It was shown that IL-4 infusion decreased the inflammatory response, as seen by a decreased lung index (1.7 in mice given F. rectivirgula and 10(3) U IL-4 and 1.31 in mice given antigen and 10(4) U IL-4 weekly versus 2.3 in mice instilled with F. rectivirgula). Interleukin-4 infusion also partially abrogated the F. rectivirgula-induced alveolitis, as seen by a decrease in cell numbers in the broncho-alveolar lavage (BAL) (1.3 x 10(5) cells in saline-instilled mice; 8.3 x 10(5) cells in mice given 10(3) U IL-4 and F. rectivirgula; 3.2 x 10(5) cells in mice given antigen and 10(4) U IL-4; 1.8 x 10(6) cells in mice given F. rectivirgula only). Also, it was apparent that IL-4 administration could partially block the appearance of the fibrosis induced by F. rectivirgula (220 micrograms of hydroxyproline/lung in challenged mice; 170 micrograms/lung in challenged mice given 10(3) U IL-4; 131 micrograms/lung in mice given antigen and 10(4) U IL-4 and c. 100 micrograms/lung in control animals). Infusion of 10(2) U IL-4 weekly had no statistical effect on any marker of inflammation.(ABSTRACT TRUNCATED AT 250 WORDS)

Entamoeba histolytica Extract and Interferon-Gamma Activation of Macrophage-Mediated Amoebicidal Function

The effect of recombinant murine interferon-gamma (IFN-gamma) and E. histolytica extract (E.h.E.) on macrophage (M phi) activation for amoebicidal activity was examined. Peritoneal macrophages were harvested from C57BL/6 and A/J mice and preincubated with IFN-gamma and/or E.h.E. It was found that amoebicidal activity could be induced in both C57BL/6 and A/J-derived macrophages by pretreatment with IFN-gamma and E.h.E. Pretreatment of the M phi with E. histolytica extract or IFN-gamma alone did not result in the activation of significant cytotoxic activity against E. histolytica trophozoites. In the presence of IFN-gamma, E.h.E. had a dose-dependent effect on the activation of M phi amoebicidal function.

Antigen-induced alveolitis: Cytokine production in a mouse model

Mice of the C57BL/6 strain were injected intraperitoneally with 108 sheep red blood cells (SRBC), then instilled intratracheally with 108 SRBC two to three weeks later. After a single intratracheal exposure, a significant cellular infiltrate occurred, composed mostly of macrophages and lymphocytes. Lymphocytes proliferated significantly in response to SRBC antigen in vitro and released interleukin-2 (IL-2). Alveolar macrophages isolated from mice challenged with SRBC released higher levels of IL-1, IL-6, and tumor necrosis factor-alpha (TNF-α) upon in vitro lipopolysaccharide (LPS) stimulation compared to unprimed, challenged mice or mice receiving intraperitoneal SRBC alone. Lymphocytes from primed mice challenged three times with SRBC proliferated significantly less in response to the antigen than mice receiving one SRBC challenge and released significant levels of interferon gamma (IFN-γ). Bronchoalveolar macrophages isolated from primed mice given three SRBC challenges released slightly higher levels of TNF-α and IL-6 in response to LPS than those from unprimed mice. After the third instillation, levels of hydroxyproline in the lungs increased significantly, indicative of a fibrotic reaction. Neutralization of IL-1 (by anti-mouse type 1 IL-1 receptor) or TNF-α resulted in the partial abrogation of the initial neutrophil influx, with some effect on the subsequent lymphocyte and macrophage influx. Blocking IL-1 or IL-2 but not TNF-α also resulted in a significant decrease in lung hydroxyproline increase, as well as lung granulomatous response and fibrosis. Overall, these results suggest that lymphoproliferation in the lungs in response to an antigen can result in fibrosis, mediated in part by IL-2 and IL-1.

Release of monokines by pulmonary macrophages following antigen challenge in sensitized guinea pigs

Guinea pigs were passively sensitized with immune serum to ovalbumin (OA), control serum, or saline. Twenty-four hours later, they inhaled aerosols of OA (2% in saline), saline, or lipopolysaccharide (LPS). Following anesthesia, bronchoalveolar lavage (BAL) was performed at 30, 60, 90 and 120 min postinhalation. Alveolar macrophages (AM) were isolated from the BAL fluid and incubated (18 h) in medium alone or with zymosan (1 mg/ml). Supernatants were collected and levels of interleukin-1 (IL-1), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-α) determined by bioassays. Unstimulated AM from animals that inhaled OA, saline, or LPS secreted similar amounts of IL-1 at 30, 60, and 90 min postinhalation. Zymosan (1 mg/ml) significantly increased IL-1 secretion by AM collected at 60 and 90 min from OA-sensitized animals that inhaled OA or saline. AM from guinea pigs sensitized to OA that inhaled OA or LPS secreted significantly increased amounts of IL-6 at 30, 60, 90, and 120 min postchallenge compared to saline sensitized controls. In all groups, AM from LPS-treated animals secreted large amounts of TNF-α at all sampling times postchallenge; AM from OA-sensitized and challenged animals secreted increasing amounts of TNF-α with time postchallenge, spontaneously and in response to zymosan. By contrast, AM from saline sensitized and challenged guinea pigs did not release detectable amounts of TNF-α spontaneously and secreted very low amounts in the presence of zymosan. These findings show that antigen challenge results in a rapid activation of AM isolated from BAL and suggest AM may initiate the development of inflammatory processes associated with antigen challenge.