Marek Rola-Pleszczynski

Leukotriene B4 induces human suppressor lymphocytes

Leukotrienes C 4 , D 4 and E 4 are the main bioactive components of slow-reacting substance of anaphylaxis, while leukotriene B 4 has proven to be a potent chemoattractant for neutrophils. We report that LTB 4 is also a potent inducer of suppressor cell activity at concentrations as low as 1 × 10 −14 M. The induced suppressor cells are T lymphocytes which do not require proliferation to exert their activity and which are sensitive to irradiation at 2000 rads. We suggest that LTB 4 may play an important role in immuno-regulation during hypersensitivity reactions.

Leukotriene B4 augments human natural cytotoxic cell activity

We have recently shown that leukotriene B4 (LTB4) activates T lymphocytes to become suppressor cells. We now report that LTB4 also augments human natural cytotoxic cell activity against target cells infected with herpes simplex virus. This activity is partially inhibited by the lipoxygenase inhibitor nordihydroguaiaretic acid and the thromboxane synthetase inhibitor OKY-1581, but is augmented by indomethacin. We suggest that LTB4 may play a role in early host defense responses during inflammatory and infectious disease processes.

Stimulation of interleukin 2 and interferon gamma production by leukotriene B4 in human lymphocyte cultures

Interleukin 2 (IL-2) and interferon-gamma (IFN) production by human lymphocytes was significantly enhanced in the presence of leukotriene B4 (LTB4) and indomethacin. Depletion of the T8+ suppressor cell subset of human T cells resulted in enhanced lymphokine production and further augmentation by LTB4. These findings suggest that LTB4 can regulate immune responses through its modulation of lymphokine production.

Asbestos-induced lung injury in the sheep model: The initial alveolitis

In order to study the cellular and biochemical changes in early asbestosis, three groups of sheep were repeatedly exposed to intratracheal instillations of either saline (controls), low doses of UICC chrysotile asbestos (LD), or high doses of the fibers (HD) until an alveolitis was observed in all HD sheep during the twelfth month of exposure. All sheep were studied bimonthly by transbronchial lung biopsy (LB), bronchoalveolar lavage (BAL), pulmonary function tests (PFT), and chest roentgenograms (CXR). While LBs of the HD sheep demonstrated large accumulations of monocyte-macrophages in the alveolar and interstitial spaces, those of controls and LD sheep did not. In BAL, there was no difference in total and differential cell counts between groups, but the BAL lymphocyte proliferative capacity was clearly depressed in all asbestos-exposed sheep. In the BAL supernatant, total proteins (mainly albumin, beta + gamma globulins) and lactate dehydrogenase were significantly elevated in the HD group only. This alveolitis was associated with a fall in vital capacity, lung compliance, diffusing capacity, and arterial PO2. Abnormalities on CXR appeared 3 months later. Thus, the cellular and biochemical features of early asbestosis are clearly distinct from those reported in idiopathic pulmonary fibrosis.

Asbestos-induced lung inflammation. Role of local macrophage-derived chemotactic factors in accumulation of neutrophils in the lungs.

In a recently developed sheep model of progressive low-dose pulmonary exposure to asbestos, initial alveolar lymphocyte and macrophage activation was followed by pulmonary neutrophilia in higher-dose exposed animals. This was observed after 18 months of exposure to intratracheal instillations of asbestos fibers and coincided with progressive alveolitis and decreased lung compliance. Alveolar macrophages from those animals were found to produce a chemotactic factor which attracted in vitro peripheral blood neutrophils. It attracted neutrophils more efficiently from higher-exposure than lower-exposure animals. Supernatants of control macrophages exposed to asbestos in vitro were also chemotactic for PMN. Similarly, bronchoalveolar lavage fluids from the animals showed chemotactic activity for peripheral blood neutrophils. It is suggested that chronic exposure to asbestos activates alveolar macrophages to produce chemotactic factors which in turn attract neutrophils from the vascular compartment and may thus contribute to the inflammatory process in asbestosis.

CCL26/eotaxin-3 is more effective to induce the migration of eosinophils of asthmatics than CCL11/eotaxin-1 and CCL24/eotaxin-2.

CCL11, CCL24, and CCL26 are chemokines involved in the recruitment of eosinophils into tissues and mainly activate CCR3. Whereas the genomic or pharmacological inhibition of CCR3 prevents the development of experimental asthma in rodents, it only impairs the recruitment of eosinophils by ∼40% in humans. As humans, but not rodents, express CCL26, we investigated the impact of CCL11, CCL24, and CCL26 on human eosinophils recruitment and evaluated the involvement of CCR3. The migration of eosinophils of healthy volunteers was similar for the three eotaxins. Eosinophils of mild asthmatics had a greater response to CCL11 and a much greater response to CCL26. Whereas all eotaxins induced the migration of eosinophil of asthmatics from 0 to 6 h, CCL26 triggered a second phase of migration between 12 and 18 h. Given that the CCR3 antagonists SB 328437 and SB 297006 inhibited the 5‐oxo‐eicosatetraenoate‐induced migration of eosinophils and that the CCR3 antagonist UCB 35625 was not specific for CCR3, CCR3 blockade was performed with the CCR3 mAb. This antibody completely blocked the effect of all eotaxins on eosinophils of healthy subjects and the effect of CCL24 on the eosinophils of asthmatics. Interestingly, CCR3 blockade did not affect the second migration phase induced by CCL26 on eosinophils of asthmatics. In conclusion, CCL26 is a more effective chemoattractant than CCL11 and CCL24 for eosinophils of asthmatics. The mechanism of this greater efficiency is not yet defined. However, these results suggest that CCL26 may play a unique and important role in the recruitment of eosinophils in persistent asthma.

Human natural cytotoxic cell activity: enhancement by leukotrienes (LT) A4, B4 and D4, but not by stereoisomers of LTB4 or HETES

Natural cytotoxic activity of human lymphocytes was significantly enhanced by incubation with LTA4, LTB4 or LTD4. In contrast, stereoisomers of LTB4 as well as 5-HETE and 12-HETE were not active. These findings suggest that leukotrienes may play an important role in host defense mechanisms.

Differential signaling pathways in platelet-activating factor-induced proliferation and interleukin-6 production by rat vascular smooth muscle cells.

Vascular smooth muscle cells (SMCs) can be induced to proliferate in response to several cytokines and growth factors, including interleukin (IL)-6. Platelet-activating factor (PAF) also has been shown to induce SMC proliferation. Because PAF can stimulate IL-6 production in monocytes, macrophages, and endothelial cells, our study was undertaken to determine whether PAF could induce IL-6 production by SMCs and to define the underlying signaling pathways. Exposure of rat aortic SMCs to picomolar concentrations of PAF resulted in enhanced production of IL-6. The effect was concentration dependent, selective for the active form of PAF, and mediated by specific PAF receptors. Pretreatment of the cells with Bordatella pertussis toxin (PTX) prevented the effect of PAF, suggesting the involvement of alpha i-type subunits of G proteins in the signal-transduction pathway. PAF-dependent IL-6 production was also prevented by inhibition of tyrosine kinases with genistein or erbstatin. Inhibition of eicosanoid production by blocking either phospholipase A2 or cyclooxygenase also abrogated the effect of PAF on IL-6 production. Moreover, inhibition of Ca2+-calmodulin activity with W7 or blocking of calcium channels with verapamil or nifedipine prevented PAF-mediated enhancement of IL-6 production. Whereas PAF-induced signal-transduction pathways leading to IL-6 production and SMC proliferation were partially common, they appeared to diverge downstream of PLA2 activation: inhibition of cyclooxygenase had no effect on proliferation, whereas augmentation of cyclic adenosine monophosphate (cAMP) levels or activation of protein kinase A inhibited proliferation, in contrast to IL-6 production. Our findings suggest a role for PAF in modulating vascular function by stimulating local production of IL-6 by SMCs and promoting their proliferation. The two effects are, however, associated with partially divergent signaling pathways and may not be causally related.

Signaling by the cysteinyl-leukotriene receptor 2. Involvement in chemokine gene transcription.

Cysteinyl-leukotrienes are involved in inflammation and act on at least two G-protein-coupled receptors, CysLT1 and CysLT2. However, the role of the CysLT2 receptor as well as its signaling remain poorly understood. Here we show that leukotriene (LT)C4 induced the production of the chemokine interleukin (IL)-8 in endothelial cells. To further study the signaling cascade involved, HEK293 cells were stably transfected with CysLT2 and used to study the transcriptional regulation of the IL-8 promoter. Stimulation of the cells with increasing concentrations of LTC4 resulted in a time- and concentration-dependent induction of IL-8 transcription and protein synthesis. Use of IL-8 promoter mutants with substitutions in their NF-κB, AP-1, or NF-IL-6 binding elements revealed an almost total requirement for NF-κB and AP-1 elements, and a lesser requirement for the NF-IL-6 element. Overexpression of dominant-negative IκBα prevented the IL-8 transactivation induced by LTC4. LTC4 stimulation induced NF-κB and AP-1 DNA binding, which involved the formation of a p50/p65 and a c-JUN·c-FOS complex, respectively. Transfection of the cells with a dominant negative (dn) form of PKCϵ prevented p65 phosphorylation, whereas dnPKCδ prevented AP-1 binding. Moreover, dnPKCδ, dnPKCϵ, and dnPKCζ prevented LTC4-induced IL-8 transcription in response to LTC4. Our data show for the first time that LTC4 can act via the CysLT2 receptor to transcriptionally activate chemokine production through induction of NF-κB and AP-1 transcription factors. These findings suggest the potential implication of CysLT2 in the inflammatory response through the modulation of chemokine gene transcription.

Expression of Platelet-Activating Factor Receptor in Human Carotid Atherosclerotic Plaques Relevance to Progression of Atherosclerosis

Background—Human monocyte-derived macrophages synthesize numerous proinflammatory and prothrombotic substances, including lipid mediators, such as platelet-activating factor (PAF), which may play a major role in the onset and perpetuation of atherosclerotic lesions. In addition, both monocytes and macrophages express PAF receptors (PAF-R). The expression of PAF-R is transcriptionally downregulated by oxidized LDL in in vitro primary cultures of monocyte/macrophages. In this study, we evaluated the expression of PAF-R in human carotid plaque tissue, in foam cells isolated from human carotid plaques, and in primary cultures of umbilical smooth muscle cells (SMCs). Methods and Results—We show that PAF-R was expressed at low levels in foam cells compared with monocyte/macrophages in plaques, as assessed by immunohistochemical staining and in situ hybridization. In addition, low levels of mRNA were also detected by RT-PCR in isolated human carotid foam cells. A prominent finding of our study was the demonstration that contractile SMCs were positive for PAF-R, and its mRNA was extracted from primary cultures of umbilical SMCs. Conclusions—As macrophages loose their inflammatory phenotype on transformation into foam cells, they may equally loose their capacity of defense against aggression. We postulate that the diminished expression of PAF-R may be deleterious in the context of plaque formation and progression. The observation that arterial SMCs of contractile phenotype express PAF-R opens new avenues concerning the migration of these cells from media to intima and atherosclerotic plaque formation.