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Leukotriene B4-loaded microspheres: a new therapeutic strategy to modulate cell activation.

Nicolete R, Rius C, Piqueras L, Jose PJ, Sorgi CA, Soares EG, Sanz MJ, Faccioli LH - BMC Immunol. (2008)

Bottom Line: In the mice cremasteric microcirculation, intraescrotal injection of 0.1 ml of LTB4-loaded MS provoked significant increases in leukocyte rolling flux, adhesion and emigration besides significant decreases in the leukocyte rolling velocity.Monocyte chemoattractant protein-1 (MCP-1) and nitric oxide (NO) productions were also increased when human umbilical vein and artery endothelial cells (HUVECs and HUAECs, respectively) were stimulated with LTB4-loaded MS.LTB4-loaded MS preserve the biological activity of the encapsulated mediator indicating their use as a new strategy to modulate cell activation, especially in the innate immune response.

View Article: PubMed Central - HTML - PubMed

Affiliation: Departamento de Análises Clínicas, Toxicológicas e Bromatológicas, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, Av, do Café s/n, 14040-903, Ribeirão Preto, São Paulo, Brasil. nicolete@fcfrp.usp.br; Cristina Rius

ABSTRACT

Background: Leukotriene B4 (LTB4) is a potent inflammatory mediator that also stimulates the immune response. In addition, it promotes polymorphonuclear leukocyte phagocytosis, chemotaxis, chemokinesis and modulates cytokines release. Regarding chemical instability of the leukotriene molecule, in the present study we assessed the immunomodulatory activities conferred by LTB4 released from microspheres (MS). A previous oil-in-water emulsion solvent extraction-evaporation method was chosen to prepare LTB4-loaded MS.

Results: In the mice cremasteric microcirculation, intraescrotal injection of 0.1 ml of LTB4-loaded MS provoked significant increases in leukocyte rolling flux, adhesion and emigration besides significant decreases in the leukocyte rolling velocity. LTB4-loaded MS also increase peroxisome proliferator-activated receptor-alpha (PPARalpha) expression by murine peritoneal macrophages and stimulate them to generate nitrite levels. Monocyte chemoattractant protein-1 (MCP-1) and nitric oxide (NO) productions were also increased when human umbilical vein and artery endothelial cells (HUVECs and HUAECs, respectively) were stimulated with LTB4-loaded MS.

Conclusion: LTB4-loaded MS preserve the biological activity of the encapsulated mediator indicating their use as a new strategy to modulate cell activation, especially in the innate immune response.

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Related in: MedlinePlus

NO production (A) and PPARα expression (B) by peritoneal macrophages after 4 hours incubation in RPMI medium. Peritoneal macrophages were incubated for 4 h with LTB4 in solution (5 × 10-8 M) with or without a specific BLT1 receptor antagonist (CP105,696). Similarly, cells were incubated with unloaded or LTB4-loaded MS (1 mg/ml) in absence or presence of CP105,696. (A) The nitrites levels were quantified by Griess reaction in the supernatants of the cells. The data are expressed as means ± the SEM (n = 3); **P < 0.01, ***P < 0.001, when compared to control (medium). ##P < 0.01, LTB4-loaded MS compared to LTB4-loaded MS + CP. (B) Western blot analysis for PPARα was performed as previously described in the Materials and Methods Section. Densitometric measurements show representative data of three separated experiments (mean ± SEM). **P < 0.01, LTB4-loaded MS + CP compared to LTB4 solution + CP. ##P < 0.01, LTB4-loaded MS + CP compared to LTB4-loaded MS.
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Figure 4: NO production (A) and PPARα expression (B) by peritoneal macrophages after 4 hours incubation in RPMI medium. Peritoneal macrophages were incubated for 4 h with LTB4 in solution (5 × 10-8 M) with or without a specific BLT1 receptor antagonist (CP105,696). Similarly, cells were incubated with unloaded or LTB4-loaded MS (1 mg/ml) in absence or presence of CP105,696. (A) The nitrites levels were quantified by Griess reaction in the supernatants of the cells. The data are expressed as means ± the SEM (n = 3); **P < 0.01, ***P < 0.001, when compared to control (medium). ##P < 0.01, LTB4-loaded MS compared to LTB4-loaded MS + CP. (B) Western blot analysis for PPARα was performed as previously described in the Materials and Methods Section. Densitometric measurements show representative data of three separated experiments (mean ± SEM). **P < 0.01, LTB4-loaded MS + CP compared to LTB4 solution + CP. ##P < 0.01, LTB4-loaded MS + CP compared to LTB4-loaded MS.

Mentions: We investigated nitrites levels produced by murine macrophages as a prediction of NO generation. High levels of nitrites were only achieved when the cells were stimulated with LTB4-loaded MS both in the presence or absence of CP 105,696 (Fig. 4A). Moreover, a further increase in nitrites production was detected when the LTB4-loaded MS were co-incubated with the BLT1 antagonist (CP 105,696) (Fig. 4A). Conversely, LTB4 in solution with or without antagonist had no effect on NO release. To assess the expression of the nuclear receptors and their possible activation under the effect of LTB4-loaded MS, we detected PPARα (molecular weight of approximately 52 kDa) in murine macrophages by Western Blot analysis. Treatment with LTB4 in solution increased PPARα expression moderately and this effect was blocked by pre-treatment of the cells with the BLT1 antagonist (CP 105,696) (Fig. 4B). However, when the cells were stimulated with LTB4-loaded MS, a further increase in PPARα expression was noted. Interestingly, a marked and significant increase in PPARα expression was also observed when the cells were stimulated with LTB4-loaded MS in the presence of the antagonist, especially when compared to that observed for the treatments with LTB4 solution + CP and LTB4-loaded MS.


Leukotriene B4-loaded microspheres: a new therapeutic strategy to modulate cell activation.

Nicolete R, Rius C, Piqueras L, Jose PJ, Sorgi CA, Soares EG, Sanz MJ, Faccioli LH - BMC Immunol. (2008)

NO production (A) and PPARα expression (B) by peritoneal macrophages after 4 hours incubation in RPMI medium. Peritoneal macrophages were incubated for 4 h with LTB4 in solution (5 × 10-8 M) with or without a specific BLT1 receptor antagonist (CP105,696). Similarly, cells were incubated with unloaded or LTB4-loaded MS (1 mg/ml) in absence or presence of CP105,696. (A) The nitrites levels were quantified by Griess reaction in the supernatants of the cells. The data are expressed as means ± the SEM (n = 3); **P < 0.01, ***P < 0.001, when compared to control (medium). ##P < 0.01, LTB4-loaded MS compared to LTB4-loaded MS + CP. (B) Western blot analysis for PPARα was performed as previously described in the Materials and Methods Section. Densitometric measurements show representative data of three separated experiments (mean ± SEM). **P < 0.01, LTB4-loaded MS + CP compared to LTB4 solution + CP. ##P < 0.01, LTB4-loaded MS + CP compared to LTB4-loaded MS.
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Related In: Results  -  Collection

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Figure 4: NO production (A) and PPARα expression (B) by peritoneal macrophages after 4 hours incubation in RPMI medium. Peritoneal macrophages were incubated for 4 h with LTB4 in solution (5 × 10-8 M) with or without a specific BLT1 receptor antagonist (CP105,696). Similarly, cells were incubated with unloaded or LTB4-loaded MS (1 mg/ml) in absence or presence of CP105,696. (A) The nitrites levels were quantified by Griess reaction in the supernatants of the cells. The data are expressed as means ± the SEM (n = 3); **P < 0.01, ***P < 0.001, when compared to control (medium). ##P < 0.01, LTB4-loaded MS compared to LTB4-loaded MS + CP. (B) Western blot analysis for PPARα was performed as previously described in the Materials and Methods Section. Densitometric measurements show representative data of three separated experiments (mean ± SEM). **P < 0.01, LTB4-loaded MS + CP compared to LTB4 solution + CP. ##P < 0.01, LTB4-loaded MS + CP compared to LTB4-loaded MS.
Mentions: We investigated nitrites levels produced by murine macrophages as a prediction of NO generation. High levels of nitrites were only achieved when the cells were stimulated with LTB4-loaded MS both in the presence or absence of CP 105,696 (Fig. 4A). Moreover, a further increase in nitrites production was detected when the LTB4-loaded MS were co-incubated with the BLT1 antagonist (CP 105,696) (Fig. 4A). Conversely, LTB4 in solution with or without antagonist had no effect on NO release. To assess the expression of the nuclear receptors and their possible activation under the effect of LTB4-loaded MS, we detected PPARα (molecular weight of approximately 52 kDa) in murine macrophages by Western Blot analysis. Treatment with LTB4 in solution increased PPARα expression moderately and this effect was blocked by pre-treatment of the cells with the BLT1 antagonist (CP 105,696) (Fig. 4B). However, when the cells were stimulated with LTB4-loaded MS, a further increase in PPARα expression was noted. Interestingly, a marked and significant increase in PPARα expression was also observed when the cells were stimulated with LTB4-loaded MS in the presence of the antagonist, especially when compared to that observed for the treatments with LTB4 solution + CP and LTB4-loaded MS.

Bottom Line: In the mice cremasteric microcirculation, intraescrotal injection of 0.1 ml of LTB4-loaded MS provoked significant increases in leukocyte rolling flux, adhesion and emigration besides significant decreases in the leukocyte rolling velocity.Monocyte chemoattractant protein-1 (MCP-1) and nitric oxide (NO) productions were also increased when human umbilical vein and artery endothelial cells (HUVECs and HUAECs, respectively) were stimulated with LTB4-loaded MS.LTB4-loaded MS preserve the biological activity of the encapsulated mediator indicating their use as a new strategy to modulate cell activation, especially in the innate immune response.

View Article: PubMed Central - HTML - PubMed

Affiliation: Departamento de Análises Clínicas, Toxicológicas e Bromatológicas, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, Av, do Café s/n, 14040-903, Ribeirão Preto, São Paulo, Brasil. nicolete@fcfrp.usp.br; Cristina Rius

ABSTRACT

Background: Leukotriene B4 (LTB4) is a potent inflammatory mediator that also stimulates the immune response. In addition, it promotes polymorphonuclear leukocyte phagocytosis, chemotaxis, chemokinesis and modulates cytokines release. Regarding chemical instability of the leukotriene molecule, in the present study we assessed the immunomodulatory activities conferred by LTB4 released from microspheres (MS). A previous oil-in-water emulsion solvent extraction-evaporation method was chosen to prepare LTB4-loaded MS.

Results: In the mice cremasteric microcirculation, intraescrotal injection of 0.1 ml of LTB4-loaded MS provoked significant increases in leukocyte rolling flux, adhesion and emigration besides significant decreases in the leukocyte rolling velocity. LTB4-loaded MS also increase peroxisome proliferator-activated receptor-alpha (PPARalpha) expression by murine peritoneal macrophages and stimulate them to generate nitrite levels. Monocyte chemoattractant protein-1 (MCP-1) and nitric oxide (NO) productions were also increased when human umbilical vein and artery endothelial cells (HUVECs and HUAECs, respectively) were stimulated with LTB4-loaded MS.

Conclusion: LTB4-loaded MS preserve the biological activity of the encapsulated mediator indicating their use as a new strategy to modulate cell activation, especially in the innate immune response.

Show MeSH
Related in: MedlinePlus