Mechanisms and Effects of Macrophage Polarization and Its Specifics in Pulmonary Environment.

Macrophages are a specific group of cells found in all body tissues. They have specific characteristics in each of the tissues that correspond to the functional needs of the specific environment. These cells are involved in a wide range of processes, both pro-inflammatory and anti-inflammatory ("wound healing"). This is due to their specific capacity for so-called polarization, a phenotypic change that is, moreover, partially reversible compared to other differentiated cells of the human body. This promises a wide range of possibilities for its influence and thus therapeutic use. In this article, we therefore review the mechanisms that cause polarization, the basic classification of polarized macrophages, their characteristic markers and the effects that accompany these phenotypic changes. Since the study of pulmonary (and among them mainly alveolar) macrophages is currently the focus of scientific interest of many researchers and these macrophages are found in very specific environments, given mainly by the extremely high partial pressure of oxygen compared to other locations, which specifically affects their behavior, we will focus our review on this group.

Hypoxia-inducible factors activator, roxadustat, increases pulmonary vascular resistance in rats.

Activators of hypoxia inducible factors (HIFs), such as roxadustat, are promising agents for anemia treatment. However, since HIFs are also involved in the regulation of the pulmonary circulation, we hypothesized that roxadustat increases pulmonary vascular resistance and vasoconstrictor reactivity. Using isolated, cell-free solution perfused rat lungs, we found perfusion pressure-flow curves to be shifted to higher pressures by 2 weeks of roxadustat treatment (10 mg/kg every other day), although not as much as by chronic hypoxic exposure. Vasoconstrictor reactivity to angiotensin II and acute hypoxic challenges was not altered by roxadustat. Since roxadustat may inhibit angiotensin-converting enzyme 2 (ACE2), we also tested a purported ACE2 activator, diminazene aceturate (DIZE, 0.1 mM). It produced paradoxical, unexplained pulmonary vasoconstriction. We conclude that the risk of serious pulmonary hypertension is not high when roxadustat is given for 14 days, but monitoring is advisable.

Hyperoxia blunts acute hypoxia- and PGF2alpha-induced pulmonary vasoconstriction in chronically hypoxic rats.

We investigated the influence of oxygenation of in vitro lung preparation on the pulmonary vascular reactivity. Small pulmonary vessels isolated from adult male Wistar rats exposed for 4 days to hypoxia (F(iO2) = 0.1, group CH) were compared with those of normoxic controls (group N). The bath in the chamber of small vessel myograph was saturated with gas mixture containing either 21% or 95% of O(2) with 5% CO(2) and we measured the reactions of vessels to acute hypoxic challenge with 0% O(2) or to PGF(2alpha). We did not observe any difference of the contractile responses between both groups when the normoxic conditions were set in the bath. When the bath oxygenation was increased to 95% O(2), the contractions induced by hypoxic challenge and PGF(2alpha) decreased in chronically hypoxic rats and did not change in normoxic controls. We hypothesize that reduced reactivity of vessels from hypoxic rats in hyperoxia results from the effect of chronic hypoxia on Ca(2+) signaling in the vascular smooth muscle, which is modulated by increased free radical production during the exposure to chronic hypoxia and further hyperoxia.

Depletion of alveolar macrophages attenuates hypoxic pulmonary hypertension but not hypoxia-induced increase in serum concentration of MCP-1.

Exposure to hypoxia, leading to hypoxic pulmonary hypertension (HPH), is associated with activation of alveolar macrophages (AM). However, it remains unclear how AM participate in this process. There are studies which imply that the AM product monocyte chemoattractant protein-1 (MCP-1) plays an important role. Thus we tested: 1. if the selective elimination of AM attenuates HPH in rats, 2. the correlation of MCP-1 plasmatic concentrations with the presence and absence of AM during exposure to hypoxia, 3. the direct influence of hypoxia on MCP-1 production in isolated AM. We found that experimental depletion of AM attenuated the chronic hypoxia-induced increase in mean pulmonary arterial pressure, but did not affect the serum MCP-1 concentrations. Furthermore, the MCP-1 production by AM in vitro was unaffected by hypoxia. Thus we conclude that AM play a significant role in the mechanism of HPH, but MCP-1 release from these cells is most likely not involved in this process. The increase of MCP-1 accompanying the development of HPH probably originates from other sources than AM.

The contractile response of isolated small pulmonary arteries induced by activated macrophages.

To test whether macrophages can play any role in hypoxic pulmonary vasoconstriction, we tested the in vitro response of rings from small pulmonary arteries to the activation of macrophages by FMLP, a substance stimulating predominantly membrane-bound NADPH oxidase. A small vessel myograph was used to measure the responses of rings from small pulmonary arteries (300-400 microm) isolated from rat lungs. Rings from 5 rats were placed into both chambers of the myograph. The vessels were stabilized for 40 min and then normalized by automatic stretching to a wall tension equivalent to the intravascular pressure 30 mm Hg. At the start of each experiment, vessels were exposed to 80 mM K+ to obtain maximal contractile response, which was used to normalize subsequent contractile responses. 2x10(6) viable macrophages, obtained by peritoneal lavage, were added into one chamber, then 5 microM FMLP was administrated to both chambers and the tension measurement was started. The hydrogen peroxide concentration produced by stimulated macrophages was measured luminometrically. The concentrations of H2O2 in specimens from chambers containing activated macrophages rose from 3.5+/-1.5 nM to 110+/-28 nM within 25 min of stimulation, while FMLP itself didn't increase the H2O2 concentration from the baseline value (4.5+/-3 nM) in samples from control chambers. After FMLP administration, the tension of the vessel rings in the presence of macrophages reached 0.23+/-0.07 of maximal contractile response, it did not change in controls. The addition of ROS scavenger 4-hydroxy-TEMPO blocked the contractile response to the activation of macrophages. We conclude that the activation of macrophages stimulates the contraction of small pulmonary arteries and that this contraction is probably mediated by reactive oxygen species.

Hypercapnia attenuates the hypoxia-induced blunting of the reactivity in chronically hypoxic rats.

Chronic hypoxia causes oxidative injury of pulmonary vessels and attenuates their reactivity to different stimuli. When combined with hypercapnia, biochemical markers of this injury are reduced but the effect of concomitant hypoxia and hypercapnia on vascular reactivity is not fully understood. This study was therefore designed to test whether hypercapnia can prevent also the hypoxia-induced loss of reactivity of pulmonary vessels. The reactivity of vessels from rats exposed either to hypoxia or hypoxia combined with hypercapnia was tested using a small vessel myograph (M 500A, Linton, Norfolk, GB). The second and third intrapulmonary branches of pulmonary arteries were isolated under a dissecting microscope from lungs of 8 control rats (group N), 6 rats exposed to hypoxia for 5 days (isobaric, 10 % O(2), group H) and 7 rats exposed to hypoxia combined with hypercapnia for 5 days (10 % O(2), 5 % CO(2), group H+CO(2)). The transmural pressure was set by automatic normalization to 30 mm Hg. The vessel size did not vary among the groups. After stabilization we challenged the vessels twice with KCl (80 mM) and once with PGF(2alpha) (0.1 mM). There were no significant differences in KCl induced contractions among the groups. The responses to PGF(2alpha) were expressed as a ratio to the maximal tension obtained by the exposure to 80 mM KCl. Contractions induced by PGF(2alpha) were markedly reduced in group H (0.07+/-0.02) and in group H+CO(2) (0.26+/-0.03) in comparison with group N (0.83+/-0.07). The vessels of group H responded to PGF(2alpha) less than those of group H+CO(2). However we observed the attenuated reactivity also in group H+CO(2) in comparison with N. Hypercapnia therefore partially blunted the hypoxia-induced loss of reactivity in pulmonary arteries. This finding supports the hypothesis that hypercapnia significantly alters the nature of lung injury induced by chronic hypoxia.

[NADPH-oxidase and the reactive oxygen species production by macrophages]. / NADPH-oxidáza a produkce reaktivních sloucenin kyslíku makrofágy.

Macrophages play an essential role not only in the defense against the infection, but are involved in many various pathological processes. They take an important part, e.g., in the development of hypoxic pulmonary hypertension (HPH). The production of reactive oxygen species (ROS) by macrophages causes the pulmonary tissue damage, which seems to play a key role in this process. This paper is focused on the NADPH-oxidase derived ROS production in alveolar macrophages and ways of its modification. NADPH-oxidase is activated via two different pathways by many stimulators. The role of the trigger can play, besides others, integrins, molecules mediating the adherence of cells. To test a role of adherence, we compared ROS production (measured as the amount of released hydrogen peroxide by the luminoldependent chemiluminescence (LDCL)) in alveolar and peritoneal macrophages. The adherence itself triggered significantly the H2O2 production only in the alveolar macrophages. Thus we suppose that the adherence is recognised as the pathological signal only in the alveolar macrophages and it can modify macrophages response to further stimuli.