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CX3CL1 (fractalkine) and TNFα production by perfused human placental lobules under normoxic and hypoxic conditions in vitro: the importance of CX3CR1 signaling.

Szukiewicz D, Kochanowski J, Mittal TK, Pyzlak M, Szewczyk G, Cendrowski K - Inflamm. Res. (2013)

Bottom Line: LPS, anti-CX3CR1 antibodies and pirfenidone were used in respective subgroups.After perfusion, CX3CR1 expression was estimated in placental tissue using quantitative immunohistochemistry, and the final results were adjusted for the mean microvascular density.Positive immunostaining for CX3CR1 corresponded to the vascular endothelium.

View Article: PubMed Central - PubMed

Affiliation: Department of General and Experimental Pathology, Second Faculty of Medicine, Medical University of Warsaw, ul. Krakowskie Przedmiescie 26/28, 00-928, Warsaw, Poland, Dszukiewicz@hotmail.com.

ABSTRACT

Objective: Inflammation and hypoxia activate the fractalkine (CX3CL1) receptor (CX3CR1)-related signaling pathway. Tumor necrosis factor alpha (TNFα) induces CX3CL1, influencing a mechanism of CX3CL1 autoregulation by CX3CR1 expression. We compared spontaneous and lipopolysaccharide (LPS)-induced CX3CL1 and TNFα production by human placenta under normoxic vs. hypoxic conditions, with respect to CX3CR1 expression and its functional status.

Methods: Placental lobules of term placentae (N = 24) were perfused extracorporeally. CX3CL1 and TNFα concentrations were measured in the perfusion fluid by ELISA. LPS, anti-CX3CR1 antibodies and pirfenidone were used in respective subgroups. After perfusion, CX3CR1 expression was estimated in placental tissue using quantitative immunohistochemistry, and the final results were adjusted for the mean microvascular density.

Results: The highest increase in CX3CL1 concentration in response to LPS was observed in hypoxia (p < 0.05). Unlike in normoxia, anti-CX3CR1 administration in hypoxia significantly reduced the LPS-evoked response. CX3CR1 expression was augmented by hypoxia and reached 260.9 ± 41 (% ±SEM) of the reference value in normoxia. Positive immunostaining for CX3CR1 corresponded to the vascular endothelium. Pirfenidone inhibited hypoxia + LPS-related increase in TNFα production and prevented the up-regulation of CX3CR1.

Conclusion: The modulatory influence of TNFα on CX3CR1 expression in hypoxia and CX3CL1/CX3CR1 interaction may serve as a compensatory mechanism to preserve or augment the pro-inflammatory course of intercellular interactions in placental endothelium.

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Immunohistochemical visualization of the receptor CX3CR1 in placental tissue at ×400 magnification: a normoxia (group IA); b hypoxia (group IIA); c pirfenidone in hypoxia (group II+). Immunostain-positive focal regions correspond to the vascular endothelium (arrowheads)
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Fig5: Immunohistochemical visualization of the receptor CX3CR1 in placental tissue at ×400 magnification: a normoxia (group IA); b hypoxia (group IIA); c pirfenidone in hypoxia (group II+). Immunostain-positive focal regions correspond to the vascular endothelium (arrowheads)

Mentions: The results pertaining to the CX3CL1 levels are shown in Fig. 3 and Table 3 (see also the experimental setup in Fig. 2). There were no statistically significant differences in the initial concentrations of CX3CL1 (initCX3CL1). The mean initCX3CL1 in group I was 99.1 ± 27 pg/ml, while group II showed a mean initCX3CL1 level of 77.8 ± 33 pg/ml. The median, rounded to the nearest whole number, for group I was 82 [95 % confidence interval (CI) 55–123] pg/ml, whereas in group II, the median was 81 (95 % CI 56–111) pg/ml (Table 3). During the 120-min observation period with specimen collections at four time-points, significant increases in the mean CX3CL1 concentration occurred in all groups studied after the addition of LPS to the perfusion fluid (Table 3; Fig. 3). Compared to group I, the highest CX3CL1 levels (p < 0.05) were measured consistently throughout the 120-min perfusion time in hypoxic group IIA. With normoxia, the neutralizing anti-CX3CR1 antibody did not affect the production of CX3CL1. Thus, the differences in the mean CX3CL1 levels between subgroups IA and IB were not statistically significant. Unlike normoxic conditions, blockade of CX3CR1 with hypoxia produced evident disturbance of LPS-induced CX3CL1 release. Despite the increase from its initial values, the mean CX3CL1 concentrations at the respective time-points were lower in group IIB (p < 0.05) than in both group IIA and group I. As shown in Fig. 4, the mean expression of CX3CR1 in the placental tissue was significantly augmented in hypoxic group IIA and reached 260.9 ± 41 (% ±SEM) of the reference value obtained in group IA. Positive immunostaining for CX3CR1 in the placenta paraffin sections was found to be localized mainly in areas that corresponded to the vascular endothelium (Fig. 5). The differences between the groups studied in mean CX3CR1 expression at the end of the adaptive phase were not significant (data not showed).Fig. 3


CX3CL1 (fractalkine) and TNFα production by perfused human placental lobules under normoxic and hypoxic conditions in vitro: the importance of CX3CR1 signaling.

Szukiewicz D, Kochanowski J, Mittal TK, Pyzlak M, Szewczyk G, Cendrowski K - Inflamm. Res. (2013)

Immunohistochemical visualization of the receptor CX3CR1 in placental tissue at ×400 magnification: a normoxia (group IA); b hypoxia (group IIA); c pirfenidone in hypoxia (group II+). Immunostain-positive focal regions correspond to the vascular endothelium (arrowheads)
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3921448&req=5

Fig5: Immunohistochemical visualization of the receptor CX3CR1 in placental tissue at ×400 magnification: a normoxia (group IA); b hypoxia (group IIA); c pirfenidone in hypoxia (group II+). Immunostain-positive focal regions correspond to the vascular endothelium (arrowheads)
Mentions: The results pertaining to the CX3CL1 levels are shown in Fig. 3 and Table 3 (see also the experimental setup in Fig. 2). There were no statistically significant differences in the initial concentrations of CX3CL1 (initCX3CL1). The mean initCX3CL1 in group I was 99.1 ± 27 pg/ml, while group II showed a mean initCX3CL1 level of 77.8 ± 33 pg/ml. The median, rounded to the nearest whole number, for group I was 82 [95 % confidence interval (CI) 55–123] pg/ml, whereas in group II, the median was 81 (95 % CI 56–111) pg/ml (Table 3). During the 120-min observation period with specimen collections at four time-points, significant increases in the mean CX3CL1 concentration occurred in all groups studied after the addition of LPS to the perfusion fluid (Table 3; Fig. 3). Compared to group I, the highest CX3CL1 levels (p < 0.05) were measured consistently throughout the 120-min perfusion time in hypoxic group IIA. With normoxia, the neutralizing anti-CX3CR1 antibody did not affect the production of CX3CL1. Thus, the differences in the mean CX3CL1 levels between subgroups IA and IB were not statistically significant. Unlike normoxic conditions, blockade of CX3CR1 with hypoxia produced evident disturbance of LPS-induced CX3CL1 release. Despite the increase from its initial values, the mean CX3CL1 concentrations at the respective time-points were lower in group IIB (p < 0.05) than in both group IIA and group I. As shown in Fig. 4, the mean expression of CX3CR1 in the placental tissue was significantly augmented in hypoxic group IIA and reached 260.9 ± 41 (% ±SEM) of the reference value obtained in group IA. Positive immunostaining for CX3CR1 in the placenta paraffin sections was found to be localized mainly in areas that corresponded to the vascular endothelium (Fig. 5). The differences between the groups studied in mean CX3CR1 expression at the end of the adaptive phase were not significant (data not showed).Fig. 3

Bottom Line: LPS, anti-CX3CR1 antibodies and pirfenidone were used in respective subgroups.After perfusion, CX3CR1 expression was estimated in placental tissue using quantitative immunohistochemistry, and the final results were adjusted for the mean microvascular density.Positive immunostaining for CX3CR1 corresponded to the vascular endothelium.

View Article: PubMed Central - PubMed

Affiliation: Department of General and Experimental Pathology, Second Faculty of Medicine, Medical University of Warsaw, ul. Krakowskie Przedmiescie 26/28, 00-928, Warsaw, Poland, Dszukiewicz@hotmail.com.

ABSTRACT

Objective: Inflammation and hypoxia activate the fractalkine (CX3CL1) receptor (CX3CR1)-related signaling pathway. Tumor necrosis factor alpha (TNFα) induces CX3CL1, influencing a mechanism of CX3CL1 autoregulation by CX3CR1 expression. We compared spontaneous and lipopolysaccharide (LPS)-induced CX3CL1 and TNFα production by human placenta under normoxic vs. hypoxic conditions, with respect to CX3CR1 expression and its functional status.

Methods: Placental lobules of term placentae (N = 24) were perfused extracorporeally. CX3CL1 and TNFα concentrations were measured in the perfusion fluid by ELISA. LPS, anti-CX3CR1 antibodies and pirfenidone were used in respective subgroups. After perfusion, CX3CR1 expression was estimated in placental tissue using quantitative immunohistochemistry, and the final results were adjusted for the mean microvascular density.

Results: The highest increase in CX3CL1 concentration in response to LPS was observed in hypoxia (p < 0.05). Unlike in normoxia, anti-CX3CR1 administration in hypoxia significantly reduced the LPS-evoked response. CX3CR1 expression was augmented by hypoxia and reached 260.9 ± 41 (% ±SEM) of the reference value in normoxia. Positive immunostaining for CX3CR1 corresponded to the vascular endothelium. Pirfenidone inhibited hypoxia + LPS-related increase in TNFα production and prevented the up-regulation of CX3CR1.

Conclusion: The modulatory influence of TNFα on CX3CR1 expression in hypoxia and CX3CL1/CX3CR1 interaction may serve as a compensatory mechanism to preserve or augment the pro-inflammatory course of intercellular interactions in placental endothelium.

Show MeSH
Related in: MedlinePlus