Photodynamic therapy mediates innate immune responses via fibroblast-macrophage interactions.

Antibacterial photodynamic therapy (PDT) has come to attract attention as an alternative therapy for drug-resistant bacteria. Recent reports revealed that antibacterial PDT induces innate immune response and stimulates abundant cytokine secretion as a part of inflammatory responses. However, the underlying mechanism how antibacterial PDT interacts with immune cells responsible for cytokine secretion has not been well outlined. In this study, we aimed to clarify the difference in gene expression and cytokine secretion between combined culture of fibroblasts and macrophages and their independent cultures. SCRC-1008, mouse fibroblast cell line and J774, mouse macrophage-like cell line were co-cultured and PDT treatments with different parameters were carried out. After various incubation periods (1-24 h), cells and culture medium were collected, and mRNA and protein levels for cytokines were measured using real-time PCR and ELISA, respectively. Our results showed that fibroblasts and macrophages interact with each other to mediate the immune response. We propose that fibroblasts initially respond to PDT by expressing Hspa1b, which regulates the NF-κB pathway via Tlr2 and Tlr4. Activation of the NF-κB pathway then results in an enhanced secretion of pro-inflammatory cytokines (TNF-α, IL-6 and IL-1ß) and neutrophil chemoattractant MIP-2 and KC from macrophages.

Tensile strength and collagen content of amniotic membrane do not change after the second trimester or during delivery.

We studied the tensile strength and collagen content of the amniotic membrane in the early and late second trimester and at term in specimens obtained after spontaneous, cesarean, and prostaglandin- and stretch-induced deliveries. No significant difference in either tensile strength or collagen content was found among any group of samples. Collagenous tissue of the amnion seems to differ constitutionally from that of the cervix and vagina and to be modulated by different mechanisms during pregnancy and delivery. It also seems to resist strong physiologic tension until it is mechanically ruptured during delivery.

[Effect of prostaglandins on collagen synthesis in rabbit ovarian follicles during the ovulatory process].

Collagen fibers in the ovarian follicle undergo drastic changes at ovulation due to the preovulatory increase of collagenolytic activities. The collagen synthesis in ovaries, however, has not been elucidated yet. To clarify the regulatory role of prostaglandins (PGs) in collagen synthesis of the follicular wall in relation to the ovulatory process, we measured prolyl hydroxylase (PH), as well as lysyl oxidase (LO) activity and the content of hydroxyproline (Hyp) in ovarian follicles of the rabbits treated by hCG, hCG/indomethacin (IM) and hCG/IM/various PGs. The experimental groups consisted of; 1) untreat control group 2) ovulatory group receiving hCG 3) non-ovulatory group given PGs 4) ovulatory group given hCG and PGs 5) group in which hCG-induced ovulation was inhibited by IM (4 mg/kg) 6) group in which IM-inhibited ovulation was recovered by PGF2 alpha (1.5 mg/kg) 7) group in which IM-inhibited ovulation was not restored by PGE1 (0.1 mg/kg) and PGE2 (0.7 mg/kg). The peak activities of PH and LO in ovarian follicles were observed at 12-13 hr after hCG injection, namely, immediately after ovulation. Significant changes of these activities after hCG administration were specific to the ovaries. PH activity in the ovaries was suppressed by the administration of IM, but LO activity was not significantly suppressed. In the hCG/IM/PGF2 alpha-treated ovulatory rabbits (Group 6), PH activity recovered to nearly the level of the hCG-treated rabbits (Group 2). By addition of PGE2, ovulation did not recover but PH activity was restored to about 70% of the hCG-treated rabbits. PGE1 did not have any effect on the reversal of ovulation-blockage or restoration of PH activity. The amount of Hyp after hCG administration tended to decrease from 6 hr to 10 hr but was significantly increased from 10 hr to 13 hr. This increase of Hyp after ovulation significantly correlated with the increase of PH and LO activities. In the hCG/IM/PGF2 alpha-treated rabbits (Group 6), the changes of Hyp were similar to those the hCG-treated rabbits (Group 2). In conclusion, collagen synthetic activity, found to be regulated by PH and LO activities in the ovarian follicles, was activated after follicle rupture, resulting in reconstruction of collagen fibers, and PGs play an important role in the ovulatory process by modifying collagen synthesis.

[Effect of PGF2 alpha on ovulation and collagenolytic activities in the rabbit treated by indomethacin].

To clarify a regulatory role of prostaglandins in collagenolytic enzyme activities of the follicular wall at ovulation, we studied whether the ovulation-blocking effect of indomethacin (IM) on hCG-stimulated rabbits could be reversed by an intravenous infusion of PGF2 alpha. The activities of BANA hydrolase and DNP peptidase were also measured by using synthetic substrates N-benzoyl-DL-arginine-2-naphthylamide HCl (BANA) and DNP-Pro-Gln-Gly-Ile-Ala-Gly-Gln-D-Arg-OH (DNP peptide), respectively, every hour during the specific preovulatory period. The ovulation-blocking effect of IM (4mg/kg) was reversed by an intravenous infusion of PGF2 alpha (1.5 mg/kg) starting at 3 hr after the hCG injection. In the control rabbits treated with hCG(100 iu) alone, these enzymatic activities were increased toward ovulation with the peak level 7-9 hr after a hCG injection and decreased significantly at 10 hr. The preovulatory increase in these enzymatic activities disappeared following the ovulation-blocking doses of indomethacin. By the addition of the PGF2 alpha infusion, the IM-induced changes in BANA hydrolase and DNP peptidase activities were returned close to the control. It is suggested that the preovulatory increases in BANA hydrolase and DNP peptidase are mandatory for ovulation and PGF2 alpha facilitates ovulation through its regulatory action on these enzymes.

[Effect of indomethacin on collagenolytic enzyme activities in rabbit ovary].

To investigate the possible participation of prostaglandins in the activation of collagenolytic enzymes of the follicular wall at ovulation, we measured the activities of neutral and acid collagenase in the rabbit ovaries at various stages of follicular development by using synthetic substrate DNP-Pro-Gln-Ile-Ala-Gly-Gln-D-Arg OH (DNP peptide) with its optimal pH 7.6 and alpha-N-benzoyl-DL-arginine-2-naphthylamide HCl (BANA) with pH 6.0, and the effect of ovulation-blocking doses of indomethacin (4 mg/kg) on DNP peptidase and BANA hydrolase activities were investigated. DNP peptidase and BANA hydrolase activities were increased toward ovulation with the highest level 7 to 9hrs after the hCG injection and then decreased significantly at 10hr. At 11hr, around the time of ovulation, the activity stayed at its low level, then rose by 13hr. Following the concomitant administration of IM with hCG, ovulation was blocked and the preovulatory increases in DNP peptidase and BANA hydrolase activities were not observed and their activities stayed at the low level until 20hr. It is suggested that collagenolytic activity for the ovulatory process started to intensiby 6hrs and ended 3hrs prior to ovulation and PGs are necessary for the enzymatic activation of DNP peptidase and BANA hydrolase.

[Collagen synthetic activity in rabbit ovary during ovulation and its blockage by indomethacin].

In order to investigate the possibility of the participation of prostaglandins (PGs) in the collagen synthetic activity during the ovulatory process, we measured prolyl hydroxylase (PH) activity and the hydroxyproline (Hyp) content of ovarian follicles in rabbits after the administration of hCG and indomethacin (IM). The minimum dose of IM (4mg/kg) effective in blocking ovulation was given simultaneously with hCG to mature female Japanese white rabbits. PH activity in the ovary reached its peak level at 13hr after the hCG injection, immediately after ovulation. This increase was not observed in other tissues such as skin, aorta or ear obtained from ovulating rabbits. In the IM-treated rabbits in which ovulation was blocked, PH activity was rather suppressed and did not show any prominent peaks, although IM did not directly suppress PH activity in the in vitro assay system. The amount of Hyp did not show any significant changes during the ovulatory process or during ovulation blockage by IM. It is suggested that PGs play an important role in controlling collagen synthetic activity of the ovary through the ovulatory process.

Effects of prolyl hydroxylase inhibition on arterial collagen synthesis and blood pressure in hypertensive rats.

Changes in arterial collagen synthesis and the effects of P-1894B, a potent inhibitor of prolyl hydroxylase, were investigated on rat hypertension, induced by deoxycorticosterone acetate (DOCA) and salt. In DOCA-salt hypertensive rats, prolyl hydroxylase activity increased significantly in the abdominal aorta and in the mesenteric artery. Incorporation of 14C-proline into the thoracic aorta, abdominal aorta and mesenteric artery was higher and the hydroxyproline content of the abdominal aorta and mesenteric artery was higher than in the control rats. Treatment with P-1894B significantly inhibited prolyl hydroxylase activity, reduced arterial collagen synthesis, but did not prevent or reverse hypertension.

[Prolyl hydroxylase activity in rabbit ovary during ovulatory process].

The disruption of connective tissue and dissociation of collagen fibers that occurs in the ovarian follicle during mammalian ovulation is reportedly induced by collagenolytic enzymes. To further analyse the metabolism of ovarian collagen during the ovulatory process, prolyl hydroxylase activity was assayed because the quantity of this enzyme provides some indication of the relative amount of collagen fibrillogenesis in connective tissues. Following the induction of ovulation by hCG, prolyl hydroxylase activity increased in both the ovarian follicle and stroma. The amount of activity reached a maximum of about 300% above the control level at 13hr after hCG, i.e., 1-3hr after the estimated time of ovulation. The pattern of enzyme activity during the ovulatory process was identical in both the follicle and the stroma. The prolyl hydroxylase activity remained at more than 200% above the control level throughout the secretory phase. It is concluded that the rabbit ovarian follicle and stroma contain prolyl hydroxylase activity, and that this enzyme activity increases significantly during the ovulatory process and luteinization.