Pouch Fluid Research Articles

Inhibitory effects of traditional Chinese medicine Shimotsu-to and its included crude fractions on adjuvant-induced chronic inflammation of mice.

Effects of a traditional Chinese medicine, Shimotsu-to (a combined prescription of cnidium rhizome, peony root, angelica root and rehmannia root), and its included crude fractions were investigated on an adjuvant-induced chronic inflammation model of mice. The aqueous extract (30, 100 and 300 mg/kg, i.p.) of Shimotsu-to reduced the carmine content, granuloma weight, inflammation cell count and pouch fluid weight in the inflammation model, respectively. The extract of Shimotsu-to without cnidium at the same doses did not produce significant changes in these four inflammatory parameters. The same doses of extracts of Shimotsu-to without peony, and without angelica, weakly reduced these parameters, except for pouch fluid weight. The extract (30, 100 and 300 mg/kg) of cnidium significantly reduced these four parameters. The same doses of peony extract reduced carmine content, granuloma weight and pouch fluid weight, but less than those of the cnidium extract. The extract of cnidium and peony at the same doses reduced in an additive manner these inflammatory parameters in their combination. These results demonstrated that the Shimotsu-to extract reduced angiogenesis, granuloma formation, inflammatory cell migration and pouch fluid exudation in the adjuvant-induced chronic inflammation model. Cnidium represented the main ingredient for producing the anti-chronic inflammatory effects of Shimotsu-to extract. Cnidium and peony exhibited additive anti-inflammatory effects in combination.

Effect of Misoprostol on Acute and Chronic Inflammation.

Prostaglandins and their fatty acid precursors are important to regulation of cell function, and immune and inflammatory responses. Prostaglandin E compounds in particular have been shown to reduce inflammation and tissue injury. We examined the ability of misoprostol, the orally active analog of Prostaglandin E1, to influence inflammation in two animal models. In the subcutaneous air pouch model, acute inflammation was induced by tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta). Misoprostol reduced pouch leukocyte counts in a dose dependent manner (33--49% v control), but did not alter fluid accumulation. The non-steroidal antiinflammatory agent, diclofenac, also reduced leukocyte counts in a dose dependent manner (28--66%) without affecting pouch fluid volume. Low dose misoprostol and low dose diclofenac used together reduced leukocyte counts by 39%, suggesting an advantageous snyergy in suppression of acute inflammation. Collagen induced arthritis (CIA) is a model of chronic inflammation. Misoprostol had no effect on the severity or course of CIA. Diclofenac reduced significantly all indices of inflammation tested, including joint swelling, number of affected joints and ability to walk. Misoprostol interfered with the antiinflammatory effect of diclofenac when the two compounds were administered together in the CIA model. These studies suggest that Misoprostol suppresses neutrophil mediated acute inflammation.

Dexamethasone inhibits the production of macrophage inflammatory protein 2 in the leukocytes in rat allergic inflammation

In the air pouch-type allergic inflammation model in rats, when the infiltrated leukocytes in the pouch fluid collected 4 h after antigen challenge were incubated, neutrophil chemotactic activity in the conditioned medium increased time-dependently. They produced neutrophil chemotactic factors, viz. leukocyte-derived neutrophil chemotactic factor (LDNCF)-2, a major component, and LDNCF-1, a minor component. When the infiltrated leukocytes were incubated in the presence of dexamethasone, neutrophil chemotactic activity in the conditioned medium decreased in a concentration-dependent manner, and production of LDNCF-2 and LDNCF-1 was inhibited. Because purified LDNCF-2 had been found to be identical with rat macrophage inflammatory protein 2 (MIP-2), effects of dexamethasone on the level of MIP-2 mRNA in the leukocytes were investigated. Using the reverse transcription-polymerase chain reaction technique, it was demonstrated that dexamethasone suppressed the level of MIP-2 mRNA in the leukocytes. These results indicate that dexamethasone inhibits MIP-2 production at the transcription level.

Dual inhibition of nitric oxide and prostaglandin production contributes to the antiinflammatory properties of nitric oxide synthase inhibitors.

We have recently put forward the hypothesis that the dual inhibition of proinflammatory nitric oxide (NO) and prostaglandins (PG) may contribute to the antiinflammatory properties of nitric oxide synthase (NOS) inhibitors. This hypothesis was tested in the present study. A rapid inflammatory response characterized by edema, high levels of nitrites (NO2-, a breakdown product of NO), PG, and cellular infiltration into a fluid exudate was induced by the administration of carrageenan into the subcutaneous rat air pouch. The time course of the induction of inducible nitric oxide synthase (iNOS) protein in the pouch tissue was found to coincide with the production of NO2-. Dexamethasone inhibited both iNOS protein expression and NO2- synthesis in the fluid exudate (IC50 = 0.16 mg/kg). Oral administration of N-iminoethyl-L-lysine (L-NIL) or NG-nitro-L-arginine methyl ester (NO2Arg) not only blocked nitrite accumulation in the pouch fluid in a dose-dependent fashion but also attenuated the elevated release of PG. Finally, carrageenan administration produced a time-dependent increase in cellular infiltration into the pouch exudate that was inhibited by dexamethasone and NOS inhibitors. At early times, i.e., 6 h, the cellular infiltrate is composed primarily of neutrophils (98%). Pretreatment with colchicine reduced both neutrophil infiltration and leukotriene B4 accumulation in the air pouch by 98% but did not affect either NO2- or PG levels. In conclusion, the major findings of this paper are that (a) selective inhibitors of iNOS are clearly antiinflammatory agents by inhibiting not only NO but also PG and cellular infiltration and (b) that neutrophils are not responsible for high levels of NO and PG produced.

Patterns of prostaglandin E 2 and leukotriene B 4 production in inflammation induced by peptidoglycan-polysaccharide polymers of bacterial cell walls

The changing levels of prostaglandin E 2 (PGE 2) and leukotriene B 4 (LTB 4) were compared over a period of 1 h to 15 days in two kinds of inflammatory responses induced in the rat air pouch by chemically similar polymers of peptidoglycan-polysaccharide (PG-PS) isolated from cell walls of group A streptococci. The smaller polymers (100 s, average mol. wt 5.3 × 10 6) induced a more severe acute response over the first 24 h, whereas, from 3 to 15 days the larger polymers (10 p, average mol. wt 5 × 10 8) induced greater chronic inflammation, as measured by pouch fluid volume, number of infiltrating cells and weight of granulation tissue. The most prominent difference between the two kinds of responses in the pattern of PGE 2 and LTB 4 were: (a) A shift between 1 to 6 h to much higher production of PGE 2 in the exudate induced by large polymers. During this time the level of LTB 4 also increased, but continued to be higher in the exudate induced by small polymers. (b) The sustained production of relatively high levels of LTB 4 and PGE 2 in the large polymer exudate at 15 days. Changes such as these indicate that this model is useful for analyzing mediators which regulate the evolution of acute into chronic inflammation.

Characterization of leukocyte-derived neutrophil chemotactic factor-2 and its possible roles in neutrophil infiltration in allergic inflammation in rats.

This study sought to clarify the responsible chemotactic factor for neutrophils in allergic inflammation in rats. When the leukocytes collected from the pouch fluid 4 h after injection of the antigen solution into the air pouch were incubated, the neutrophil chemotactic activity in the conditioned medium increased time-dependently with higher levels for the leukocytes from the immunized rats than the nonimmunized ones. The chemotactic activity did not result from cytokine-induced neutrophil chemoattractant (CINC) because CINC concentrations in the conditioned medium were low. Neutrophil chemotactic factors in the conditioned medium were separated by isoelectric focusing into two factors, leukocyte-derived neutrophil chemotactic factor (LDNCF)-1 and LDNCF-2. The activity of LDNCF-2 was more than 75% of the total chemotactic activity in the conditioned medium. LDNCF-2 was purified by gel chromatography and reverse-phase HPLC. The N-terminal amino acid sequence (1-20) of the purified LDNCF-2 was identical to the 32-51 amino acid sequence of the pro-form of rat macrophage inflammatory protein (MIP)-2. Higher levels of MIP-2 mRNA in the leukocytes from the immunized rats than that from the non-immunized rats were proved by the reverse transcription-polymerase chain reaction. In vivo, concentrations of CINC in the pouch fluid were low, and did not represent the chemotactic activity in the pouch fluid. These results suggest that LDNCF-2 (MIP-2) is an important chemotactic factor for neutrophils in the allergic inflammation in rats.

Leukocyte-derived neutrophil chemotactic factor-2 produced by infiltrated leukocytes in allergic inflammation model in rats is macrophage inflammatory protein-2.

In the air pouch-type allergic inflammation model in rats, leukocytes collected from the pouch fluid 4 h after the antigen challenge produced proteinaceous chemotactic factors for neutrophils. The leukocytes from the immunized rats produced significantly higher amount of the chemotactic factors than that from the non-immunized rats. The major chemotactic factor, leukocyte-derived neutrophil chemotactic factor (LDNCF)-2, was purified and found to be identical with rat macrophage inflammatory protein (MIP)-2 by N-terminal amino acid sequence analysis. Expression of MIP-2 mRNA was higher in the leukocytes from the immunized rats than that from the non-immunized rats. Possible roles of LDNCF-2 (MIP-2) in neutrophil infiltration in the allergic inflammation is discussed.

Possible roles of protein kinases in neutrophil chemotactic factor production by leucocytes in allergic inflammation in rats.

1. In an air pouch-type allergic inflammation model in rats, leucocytes that had infiltrated into the pouch fluid collected 4 h after the antigen challenge produced proteinaceous chemotactic factors for neutrophils when they were incubated in the medium. 2. To clarify the mechanism of activation of the infiltrated leucocytes in producing these factors, the effects of protein kinase inhibitors on neutrophil chemotactic factor production were examined. 3. When the infiltrated leucocytes were incubated for 4 h in medium containing the non-selective protein kinase inhibitor K-252a (1-100 ng ml-1, 2.14-214 nM), the tyrosine kinase inhibitor genistein (1-50 micrograms ml-1, 3.7-185 microM), and the more selective protein kinase C inhibitor H-7 (5-100 micrograms ml-1, 13.7-274 microM); neutrophil chemotactic activity in the conditioned medium was decreased in a concentration-dependent manner, but the adenosine 3':5'-cyclic monophosphate (cAMP)-dependent protein kinase inhibitor H-89 (1-1000 ng ml-1, 2.24-2240 nM) showed no effect. 4. Isoelectric focusing of the conditioned medium revealed that the leucocytes produced two neutrophil chemotactic factors, leucocyte-derived neutrophil chemotactic factor (LDNCF) 1 and LDNCF-2. Treatment of the leucocytes with K-252a, genistein, and H-7, but not H-89, inhibited production of both LDNCF-1 and LDNCF-2. 5. These results suggest that activation of tyrosine kinase and protein kinase C, but not cAMP-dependent protein kinase, is responsible for the production of LDNCF-1 and LDNCF-2. 6. The steroidal anti-inflammatory drug dexamethasone and the protein synthesis inhibitor cycloheximide inhibited neutrophil chemotactic factor production in a concentration-dependent manner. Time-course experiments showed that the inhibitory effect by dexamethasone was apparent even 30 min after the incubation.7. Mechanism for inhibiting the production of LDNCF-1 and LDNCF-2 by dexamethasone is also discussed.

Pharmacological analysis of neutrophil chemotactic factor production by leucocytes and roles of PAF in allergic inflammation in rats.

1. The role of platelet activating factor (PAF) in neutrophil infiltration in air pouch type allergic inflammation in rats was investigated. 2. Neutrophil infiltration into the pouch fluid 8 h after injection of the antigen (azobenzenearsonate-conjugated acetyl bovine serum albumin) solution into the air pouch of immunized rats was inhibited dose-dependently by treatment with PAF antagonists (CV-3988, L-652,731 and Y-24,180) in parallel with the decrease in neutrophil chemotactic activity in the pouch fluid. 3. Four hours after injection of the antigen solution into the air pouch of immunized and non-immunized rats, there was no significant difference between the two groups in the number of total leucocytes, neutrophils, mononuclear cells and eosinophils in the pouch fluid. However, when the infiltrated leucocytes were incubated in medium, chemotactic factor production by leucocytes from immunized rats was greater than that from non-immunized rats. 4. When leucocytes from non-immunized rats were preincubated for various periods in the medium containing 10 or 50 nM of PAF, washed, and further incubated in the medium containing no PAF, chemotactic factor production was not stimulated. 5. The increase in the chemotactic activity in the conditioned medium was not suppressed by the 5-lipoxygenase inhibitor, AA861. In addition, the chemotactic activity in the conditioned medium was not inhibited by the PAF antagonists. 6. Incubation of the infiltrated leucocytes in the medium containing the protein synthesis inhibitor, cycloheximide, inhibited chemotactic factor production in a concentration-dependent manner in parallel with the decrease in uptake of [3H]-leucine into the acid-insoluble fraction of leucocytes. 7. Separation of the chemotactic activity in the conditioned medium by isoelectric focusing revealed that the leucocyte infiltrated into the pouch fluid produce two kinds of factors, viz. leucocyte-derived neutrophil chemotactic factor-i (LDNCF-1) and LDNCF-2 of which pI values are 4-5 and above 8,respectively.8. The results indicate that PAF has no significant role in leucocyte activation to produce chemotactic factors, and that neutrophil chemotactic factors produced by infiltrated leucocytes are not PAF or leukotriene B4 but are produced through a protein synthesis mechanism.9. The mechanism of action of PAF antagonists on neutrophil infiltration into the inflammatory locus is discussed.

血管内皮細胞を介する好中球遊走における血小板活性化因子の役割解析

In an air pouch type allergic inflammation model in rats, PAF antagonists inhibit neutrophil migration into the pouch fluid although levels of PAF in the pouch fluid are less than the detectable amount. To clarify roles of PAF in neutrophil migration, effects of PAF antagonists (CV-6209, L-652, 731, Y-24180) on transendothelial migration of neutrophils were examined in vitro. Histamine and thrombin stimulated LTB4-induced transmigration of neutrophils through the confluent monolayer of human umbilical vein endothelial cells (HUVEC) . Treatment with PAF antagonists selectively inhibited the histamine- and thrombin-induced transendothelial migration, suggesting that PAF plays some significant roles in endothelial cell recognition by neutrophils.Treatment of HUVEC with TNF-α or IL-1/β induced neutrophil adherence to HUVEC monolayer and expression of adhesion molecules, such as ICAM-1, ELAM-1 or GMP-140. However, PAF antagonists showed no effect on the cytokine-induced neutrophil adherence to HUVEC nor the expression of these adhesion molecules. Furthermore, histamine- and thrombin-induced neutrophil adherence to HUVEC was inhibited by PAF antagonists without altering the expression of these adhesion molecules.Transendothelial migration of neutrophils was not inhibited by antibodies to GMP-140 and ELAM-1, but was completely inhibited by an antibody to LFA-1/β. An antibody to ICAM-1 partially inhibited the transendothelial migration of neutrophils.These results suggest that PAF produced by HUVEC plays important roles in signaling for neutrophil adherence to endothelial cells without affecting the expression of these adhesion molecules.

The effects of calcium pentosan polysulfate on inflammatory mediators in the rat air-pouch model of inflammation

A new calcium-chelated derivative of pentosan polysulfate (CaPPS) is currently under investigation as a potential disease-modifying antiarthritic agent. In the present study the effects of CaPPS on edema production, cellular infiltration, and activity of inflammatory mediators (prostaglandin E 2 [PGE 2], interleukin [IL]-1β, IL-6, and tumor necrosis factor [TNF]-α) using the rat subcutaneous air-pouch model of inflammation are reported. CaPPS or saline was injected subcutaneously daily for 7 days (study days 8 to 14) at concentrations of 0.5 to 10 mg/kg of body weight into the abdomen of rats with preformed noninflamed or peptone-inflamed dorsal pouches. The pouch fluid volume, white cell count, differential white cell count, PGE 2, TNF-α, IL-6, and IL-1β levels were determined using standard methods after the rats were sacrificed on day 15. In saline-treated control animals, injection of peptone into rat air pouches provoked a large influx of fluid and white cells, with a concomitant increase in levels of PGE 2, TNF-α, IL-6, and IL-1β. Although subcutaneously administered CaPPS had no effect on the accumulation of fluid, white cell numbers, or PGE 2 levels within the pouch, there was a 38% reduction in TNF-α activity in the presence of 2.5 to 10 mg/kg body weight of drug ( P < 0.05). In contrast, IL-6 activity increased 21% to 47% compared with saline-treated control rats ( P < 0.05) when CaPPS concentrations of 0.5 to 10 mg/kg body weight were used. The levels of IL-1β in pouch fluid remained the same as saline-treated rats over this dose range. These findings indicated that CaPPS could elicit specific effects on cytokine production by leukocytes and synovial cells. It is proposed that the reduction by CaPPS in TNF-α levels in inflamed rat air pouches, combined with the ability of CaPPS to elevate IL-6, may enhance the known chondroprotective activity of this drug.

Changes in the proteins coating monosodium urate crystals during active and subsiding inflammation. Immunogold studies of synovial fluid from patients with gout and of fluid obtained using the rat subcutaneous air pouch model.

In this in vivo study, we investigated changes in the proteins that coat monosodium urate (MSU) crystals in human synovial fluid samples and rat air pouch fluid samples obtained sequentially during periods of active and resolving inflammation, in order to evaluate whether in vivo findings are consistent with hypotheses on roles of protein coating based on in vitro findings. Crystals from patients with gout were isolated from joint fluids with acute inflammation, and subsequently from the same joints at the time inflammation was resolving. Crystals were also obtained using the rat subcutaneous air pouch model. Immunogold was used to label proteins coating MSU crystals, for light microscopy (LM) and transmission electron microscopy (TEM) studies. Dense immunogold-silver labeling for IgG was observed under LM on crystals from fluid with acute inflammation, whereas other proteins (apolipoproteins [Apo], fibronectin, fibrinogen, albumin) were not labeled significantly. Apo B became strongly positive on crystals as the inflammation subsided, whereas other proteins were only weakly positive and IgG became absent or weakly positive. Quantitative TEM evaluation confirmed the LM observations. This study provides the first in vivo evidence supporting the notion derived from previous in vitro studies that proteins coating MSU crystals change as inflammation evolves. Protein coatings may play an important role in the self-limited nature of gouty inflammation. IgG coating MSU crystals may enhance the inflammation. As the inflammation subsides, Apo B could displace the IgG by competitively coating sites on crystals and could contribute in part to the resolution of the acute gouty arthritis.

Human Colostrum Has Anti-inflammatory Activity in a Rat Subcutaneous Air Pouch Model of Inflammation

An animal model was used to examine the effect of human colostrum on an acute inflammatory process in vivo. Subcutaneous air pouches on the backs of outbred rats were injected with carrageenan as an inflammatory challenge, normal saline, pooled aqueous human colostrum, or carrageenan plus colostrum concurrently. Oral dexamethasone or indomethacin was administered to some animals before and during challenge as anti-inflammatory agents. Polymorphonuclear leukocyte (PMN) counts in pouch fluid were determined 6 h postchallenge. Carrageenan challenge resulted in a significant acute inflammatory response [48.8 +/- 4.9 x 10(6) PMN/pouch (mean +/- SEM, n = 46)] compared with normal saline controls [0.9 +/- 0.2 x 10(6) (n = 31, p < 0.001 versus carrageenan)] or with colostrum [4.3 +/- 0.8 x 10(6) PMN/pouch (n = 11, p < 0.001 versus carrageenan)]. The concurrent injection of colostrum plus carrageenan challenge significantly reduced the PMN response compared with carrageenan alone [18.8 +/- 2.9 versus 48.8 +/- 4.9 x 10(6) PMN/pouch (carrageenan plus colostrum versus carrageenan, n = 41 versus 46, p < 0.001)]. This degree of suppression of PMN influx was not significantly different from that seen with indomethacin treatment but was significantly more than that seen with dexamethasone treatment. The decreases in PMN counts observed most likely reflect suppression of the acute inflammatory response because a significant amount of PMN lysis in colostrum was not observed in vitro and the accumulation of PMN granule contents was not seen in pouch fluid from colostrum-treated animals in vivo. These data directly demonstrate for the first time that human colostrum has a biologically significant effect on the inflammatory process in vivo.

Pentosan polysulphate and glycosaminoglycan polysulphate stimulate the synthesis of hyaluronan in vivo.

Pentosan polysulphate (PPS) and glycosaminoglycan polysulphate (GAGPS) were examined for their ability to alter hyaluronan synthesis in vivo. The inflamed rat subcutaneous air pouch model was used for the study. PPS or GAGPS injected into the air pouch at a dose of 2.5 mg/kg daily for 7 days resulted in higher molecular weight hyaluronan in the pouch fluid compared with control non-drug-treated pouch fluid. The quantity of the hyaluronan was increased by PPS, but not by GAGPS. We concluded that both drugs could be beneficial in the treatment of inflammatory arthritides in which a decrease in normal synovial hyaluronan concentration and molecular weight occurs.

慢性期炎症しん出液中に存在するヒスタミン産生増大因子―ＩＩの解析

Employing the air pouch-type allergic inflammation model in rats, effects of inflammatory exudate on histamine production by bone marrow cells were examined in order to clarify the mechanism of histamine production at the site of allergic inflammation. The pouch fluid collected 8 h after the induction of allergic inflammation (the acute phase) increased histamine production by bone marrow cells. On the other hand, the pouch fluid collected 5 days after the antigen challenge (the chronic phase) showed no activity by itself, but enhanced the activity by the pouch fluid collected at the acute phase. These activities were due to proteinous factors that were produced at the acute phase and at the chronic phase, named histamine-production-increasing factor (HPIF) -I and HPIF-II, respectively. HPIF-I increased histidine decarboxylase (HDC) activity in bone marrow cells but HPIF-II did not. However, HPIF-II enhanced HDC activity stimulated by HPIF-I. The cooperative effect of HPIF-I and HPIF-II on histamine production was also observed in vivo. Isoelectric point and molecular weight of HPIF-II were estimated to be 7 to 8 and about 100 kD, respectively.These results indicate that histamine production at the site of allergic inflammation might be regulated by at least two kinds of proteinous factors, HPIF-I and HPIF-II.

Combined effects of Japanese‐Sino medicine ‘Kakkon‐to‐ka‐senkyu‐shin'i’ and its related combinations and component drugs on adjuvant‐induced inflammation in mice

AbstractKakkon‐to‐ka‐senkyu‐shin'i (KSS), a Japanese‐Sino medicine consists of nine crude drugs: pueraria, ephedra, cnidium, magnolia, cassia, peony, liquorice, ginger and jujube. It has long been used clinically in the treatment of nasal inflammation. The antiinflammatory effects of KSS, its related combinations, and component crude drugs were investigated on adjuvant‐induced air pouch granuloma in mice. KSS was effective in inhibiting the inflammatory process of pouch fluid exudation, inflammatory cell migration and angiogenesis. Antiangiogenic effects were additive between (1) Keishi‐to and the combination of pueraria + ephedra + cnidium + magnolia in KSS, (2) Keishi‐to and the combination of pueraria + ephedra in Kakkon‐to, and (3) cnidium and magnolia in the combination of cnidium + magnolia. Inhibitory effects on inflammatory cell migration were also additive between (4) cnidium and magnolia in the combination of cnidium + magnolia, and were potentiative between (5) Kakkon‐to and the combination of cnidium + magnolia in KSS, (6) (pueraria+ephedra) and (cnidium + magnolia) in the combination of pueraria + ephedra + cnidium + magnolia, and (7) pueraria and ephedra in the combination of pueraria + ephedra. The main component crude drugs and combination in KSS for antiinflammation were magnolia and pueraria for angiogenesis, magnolia and ephedra for pouch fluid exudation, and pueraria + ephedra + cnidium + magnolia for inflammatory cell migration.

Induction of neutrophil infiltration by rat chemotactic cytokine (CINC) and its inhibition by dexamethasone in rats

In vivo effects of cytokine-induced neutrophil chemotactic factor (CINC) derived from rats on neutrophil infiltration were investigated using an air-pouch-type inflammation model in rats, and effects of dexamethasone on neutrophil infiltration induced by CINC was also examined in order to gain further insight into the mechanism of antiinflammatory activity of glucocorticoids. Injection of CINC into the air pouch made on the dorsum of rats induced a marked infiltration of neutrophils into the pouch fluid but not mononuclear cells and eosinophils during a 30-min interval after the injection. Maximum effect was induced at a dose of 1.4 micrograms/pouch. Treatment with dexamethasone 3 h before the injection of CINC suppressed the neutrophil infiltration in a dose-dependent manner, but no complete inhibition was observed. CINC injection into the air pouch of rats that had been sacrificed by bleeding in order to minimize neutrophil infiltration from blood stream also stimulated neutrophil infiltration into the pouch fluid when the carcass was incubated at 37 degrees C for 30 min, but the number of infiltrated neutrophils was about 35% of CINC-induced neutrophil infiltration in intact rats. CINC-induced neutrophil infiltration in the carcass, which is supposed to be a reflection of neutrophil migration from extravascular space in subcutaneous tissues to pouch fluid, was not inhibited by dexamethasone treatment. Therefore, the inhibition of neutrophil infiltration by dexamethasone might be due to inhibition of the extravasation of peripheral neutrophils but not due to inhibition of neutrophil chemotaxis from subcutaneous extravascular space to pouch fluid. These findings suggest that clinical effects of steroidal antiinflammatory drugs on neutrophil infiltration in inflammatory disease is partly due to inhibition of neutrophil extravasation induced by preformed neutrophil chemotactic factors in the inflammatory site.

Aloe vera and the inflamed synovial pouch model.

Administration of air under the skin produced a pouch wall that closely resembled a synovium in that the inner lining was made up of macrophages and fibroblasts. Administration of 1% carrageenan directly into the 7-day-old air pouch produced an inflammation characterized by an increased number of mast cells in pouch fluid as well as an increase in wall vascularity. A punch biopsy weight of the pouch wall did not reveal an increase in 1% carrageenan-treated animals. However, a 10% Aloe vera treatment of carrageenan-inflamed synovial pouches reduced the vascularity 50% and the number of mast cells in synovial fluid 48%. The pouch wall punch biopsy weight was increased by A. vera, which was verified by histologic examination of the inner synovial lining. Aloe vera stimulated the synovial-like membrane, as evidenced by an increased number of fibroblasts, suggesting that A. vera stimulated fibroblasts for growth and repair of the synovial model. The synovial air pouch can be used to study simultaneously the acute anti-inflammatory and fibroblast stimulating activities of A. vera.

Changes in the levels of rat interleukin 8/CINC and gelatinase in the exudate of carrageenin-induced inflammation in rats.

A sensitive enzyme-linked immunosorbent assay (ELISA) for rat interleukin 8/cytokine-induced neutrophil chemoattractant (CINC) has been established by using biotin-conjugated anti-CINC rabbit IgG. The biotin-streptavidin sandwich ELISA detected CINC at concentrations from 3 pg/ml up to 30 ng/ml. The concentration of CINC in the pouch fluid (exudate) of rat carrageenin-induced inflammation was measured by the ELISA. After a time lag of about 2 h, neutrophils steadily accumulated in the carrageenin/air-pouch until 8 h. Similarly, the CINC level of exudate increased after about a 2-h lag, and reached a maximum (134 ng/ml) at 8 h, and thereafter decreased to a negligible concentration at 24 h after carrageenin injection. In association with the rise in CINC level, the concentration of exudate 96-kDa gelatinase corresponding to neutrophil gelatinase/type IV collagenase increased with time. The results suggest that CINC contributes, at least in part, to the neutrophil migration into the inflammatory lesion of the carrageenin-induced inflammation in rats.

Pharmacological studies on 3-formylamino-7-methylsulfonylamino-6-phenoxy-4H-1-benzopyran-4-one (T-614), a novel antiinflammatory agent. 3rd communication: the involvement of bradykinin in its analgesic actions.

In order to elucidate the analgesic mechanism of 3-formylamino-7-methylsulfonylamino-6-phenoxy-4H-1-benzopyran-4-on e (T-614), its effects on the kinin-forming system were examined both in vivo and in vitro. T-614, at doses more than 10 mg/kg p.o., exhibited a significant inhibitory effect on the increased levels of bradykinin released into the pouch fluid of kaolin-induced inflammation in rats. In the kaolin-induced writhing response in mice, which is shown to be mainly dependent on the action of bradykinin, T-614 reduced not only the writhing frequency but also the peritoneal levels of bradykinin in a dose-dependent manner. Whereas, in the zymosan-induced writhing response in which prostaglandin I2 (PGI2) is shown to be an important mediator, it did not exert an obvious inhibition on either writhing responses or peritoneal PGI2 levels at a highest dose of 100 mg/kg. T-614 did not inhibit the activities of serine proteases, such as trypsin, thrombin, kallikrein and plasmin. Furthermore, it did not affect the kinin-forming enzymes of rat plasma in vitro. The above results suggest that the analgesic effects of T-614 may be partly mediated by the inhibition of bradykinin release in the local inflamed tissue.