Carrageenin-induced Granuloma Research Articles

Simultaneous generation of nitric oxide and superoxide by inflammatory cells in rats

It has recently been shown that peroxynitrite anion is a powerful oxidant that can initiate lipid peroxidation. As this oxidant is the product of the reaction between nitric oxide and superoxide, we have studied whether cells isolated from an inflammatory exudate can release both radicals simultaneously under physiological conditions. The carrageenin-induced granuloma model in rats was used. Cells from the inflammatory pouch were stimulated with opsonized zymosan in the absence or in the presence of exogenous l-arginine. Nitric oxide production without exogenous l-arginine was detectable after 15 min (0.29 nmol NO 2 −) and increased with time (1.65 nmol NO 2 − at 4 h). When nitrite released from cells was expressed as a rate a burst was shown in the first few minutes. Between 0 and 15 min, cells produced NO 2 − at the following rates: 20 pmol NO 2 −/1 × 10 6 cells/min without exogenous l-arginine and 83 pmol NO 2 −/1 × 10 6 cells/min with exogenous l-arginine. Production was further stimulated with opsonized zymosan (92 pmol NO 2 −/1 × 10 6 cells/min), and inhibited by L-NMMA and L-NIO. The production of superoxide increased for up to 2 h and then stabilized. A significant increase in nitrite was observed in the presence of SOD, whereas L-NIO increased superoxide generation. These results suggest that peroxynitrite anion may be formed by inflammatory cells.

Cellular Source of Collagenase and TIMP-1 in Carrageenin-Induced Granuloma

We have examined the occurrence and cellular localization of interstitial collagenase and TIMP-1 mRNAs in a model of granuloma induced by carrageenin in guinea pigs. Granulomas were studied at 4, 7, 10, and 14 days after carrageenin injury using a combined protocol for in situ hybridization and immunofluorescence. Antivimentin monoclonal antibody was used to identify fibroblasts. Avidin-FITC and Texas red horse antimouse IgG were employed for detection of probes and antibody, respectively. Our results showed that during the extracellular matrix deposit phase (4 and 7 days), interstitial collagenase and TIMP-1 mRNAs were expressed only by fibroblasts as demonstrated by the colocalization of mRNA and vimentin. By contrast, during the initiation of the resorptive phase (10 and 14 days), fibroblasts and vimentin-negative cells, probably macrophages, expressed collagenase and TIMP-1. This study suggests that fibroblasts are the cell type expressing interstitial collagenase and TIMP-1 mRNA during all phases of the evolution of carrageenin granuloma and that macrophages, by contrast, express the mRNA for the enzyme and the inhibitor exclusively in the degradative phase.

Collagenase-inhibitory activity in deposit and resorption phases of guinea pig carrageenin granuloma.

The levels of collagenase inhibitor, both free and bound to metalloproteinases, were evaluated at 7 days [deposit phase (DP)] and 14 days [resorptive phase (RP)] of evolution of the subcutaneous carrageenin-induced granuloma in the guinea pig. The level of free collagenase inhibitor was considerably higher in the supernatant of DP granulomas (7.95 +/- 1.53 U/mg protein) as compared to that of RP granulomas (2.53 +/- 0.41 U/mg protein). When the samples were heated at acid pH to release the inhibitor from metalloproteinase-inhibitor complexes, free inhibitor was recovered in both phases. However, the units of recovered collagenase inhibitor were several fold higher in all RP granulomas in comparison with DP granulomas (6.88 +/- 2.46 vs 1.5 +/- 0.53). Therefore, DP and RP tissues exhibited similar total amount of tissue inhibitor. By HPLC, collagenase inhibitor activity was localized in a fraction consistent with the size of TIMP. These results suggest a different balance of collagenase and collagenase inhibitor during the evolution of the granuloma; an excess of inhibitor over metalloproteinases appears to predominate during the phase of collagen accumulation contrasting with an inverse situation when the granuloma is healing.

Involvement of glucocorticoid in insulin-induced angiogenesis of adjuvant pouch granuloma in diabetic mice.

The effects of insulin and glucocorticoid on granuloma formation and angiogenesis were studied using an adjuvant pouch in mice and a carrageenin pouch in rats. Carrageenin-induced granuloma formation was suppressed in the diabetic state induced by alloxan in rats. The suppression was restored by adrenalectomy. Corticosterone counteracted the restoration, whereas epinephrine did not, suggesting that the effects of adrenalectomy are due to the lack of adrenocortical hormones rather than epinephrine. The blood corticosterone levels in alloxan mice increased, following the increase of glucose level, to more than 20 micrograms/dl after 4 weeks. The increase of corticosterone levels disappeared after adrenalectomy. In the mouse adjuvant pouch, corticosterone dose-dependently decreased granuloma formation and angiogenesis. The values of these two parameters obtained with a dose of 20 micrograms/pouch corticosterone agreed with those of the serum levels in diabetic mice. Insulin dose-dependently reversed the suppressed angiogenesis of 20 micrograms corticosterone-treated mice and the dose-response curve approximated the curve of alloxan-treated mice. The effects of diabetes were concluded to involve insulin deficiency as well as glucocorticoid enhancement and the counteraction between the two may control granuloma formation and angiogenesis.

Linoleic and dihomo-gamma-linolenic acids modulate granuloma growth and granuloma macrophage eicosanoid release.

We have already demonstrated that arachidonic acid (AA) inhibits carrageenin-induced granuloma growth in vivo and that this effect is related to an increase in prostaglandin E2 formation. As prostaglandin E1 has been shown to be more effective in inhibiting granuloma growth than prostaglandin E2 we investigated the effect of linoleic acid (LA) (18:2 omega 6) and dihomo-gamma-linolenic acid (DHGLA) (20:3 omega 6), potential precursors of prostaglandin E1, in this model. LA and DHGLA inhibited the development of carrageenin-induced granulomas in the rat when injected locally. Both fatty acids (FA) stimulated the release of prostaglandin (PG) E1 from granuloma macrophages (M phi) in vitro, DHGLA being most effective. LA had little effect on the release of PGE2, 6 keto PGF1 alpha, the stable product of prostacyclin (PGI2) or thromboxane (Tx) B2, the stable metabolite of TxA2. DHGLA had no effect on the release of 6 keto PGF1 alpha, but inhibited PGE2 and, to a lesser extent, TxB2 synthesis.

Glucocorticoid receptors in carrageenin-induced granuloma in rats: Activation to dexamethasone-binding form by sulfhydryl compounds

The specific binding of glucocorticoids to the cytoplasmic receptor and nuclei in granuloma tissue was studied in in vitro experiments. Although 105,000 g supernatants (cytosols) from liver and thymus homogenates indicate high affinity binding to both of cortisol and dexamethasone, granuloma cytosol was apparently lacking of the dexamethasone-binding capacity. However, a definite capacity to bind dexamethasone was demonstrated by the cytosol and nuclei of the granuloma tissue when the tissue was minced and subjected to the incubation with the steroid. Intracellular transport of dexamethasone to the nucleus via cytosol in the incubation of the minced tissue was indicated to be a function of temperature and time. Demonstration of dexamethasone binding by the cytosol from the granuloma homogenate was achieved by adding sulfhydryl-protecting agents, such as dithiothreitol, glutathione and 2-mercaptoethanol to the medium for homogenization and incubation. Treatment of cytosols from the liver and the granuloma with 5,5'-dithio-bis(2-nitro-benzoic acid) (DTNB) severely damaged their glucocorticoid-binding ability. The deteriorating effect of DTNB was antagonized by the above sulfhydryl agents. It is therefore concluded that glucocorticoid receptor, as demonstrated by dexamethasone-binding capacity. of the carrageenin-induced granuloma tissue involves active sulfhydryl group(s) which is sensitive to oxidizing agents.

Prostaglandin E 2 elevation of cyclic-AMP in granuloma macrophages at various stages of inflammation: Relevance to anti-inflammatory and immunomodulatory functions

In the carrageenin-induced granuloma of rats the inflammatory tissue growth and macrophage invasion on the one hand and the cyclic-AMP content of the macrophages on the other, display opposite directional changes. Macrophages, isolated from this tissue at different stages of inflammation, were used to examine the effect of prostaglandin E 2 on intracellular levels of c-AMP. It appears that during infiltration of the macrophages into the inflammatory tissue, the sensitivity of adenylate cyclase to activation by PGE 2 increases. Arguments are presented that these observations made in vitro , are in direct relevance to the previously described anti-inflammatory effect of PGE on granuloma tissue in vivo .

Macrophages as targets of inhibitory effects of E-type prostaglandins in immune-related inflammation.

The anti-inflammatory effects of E-prostaglandins (PGE) are attracting interest because they are mediated through actions on cells which are also targets for the putative immunomodulator functions of PGE. In the majority of experimental inflammatory conditions, in which inhibitory effects of PGE have been demonstrated, a variety of immunocytes are implicated, including diverse lymphocyte populations. The inhibitory effects of PGE, however, are also readily observable on the tissue component of the carrageenin-induced granuloma (an immune-related inflammatory model) in which activated macrophages, but not lymphocytes participate. Granuloma-derived macrophages are particularly interesting cells for the study of responsiveness to PGE, because the results obtained on such cells in vitro are directly related to the anti-inflammatory effects of PGE on the macrophage phase of the granuloma in vivo. Such combined studies have revealed differences between the responsiveness of granuloma macrophages to PGE2 and prostacyclin, while with elicited peritoneal macrophages such a difference could not be observed. The adenylate cyclase in granuloma macrophages appears to be unusually sensitive to activation by PGE2, but insensitive to prostacyclin.

Cellular origin of cathepsin B in carrageenin-induced granuloma tissues in rats.

Cathepsin B isolated from carrageenin-induced granuloma tissue was separated into two peaks (one large and one small) on CM-cellulose column chromatography. The cathepsin B activities from the peritoneal macrophages and polymorphonuclear leukocytes were eluted at the positions identical to those of the larger (P-D and the smaller (P-II) peaks, respectively. Similarly, the cathepsin B activities in the P-I and P-II fractions from granuloma tissue corresponded to those from macrophages and leukocytes, respectively, in isoelectric focusing in polyacrylamide gel. Both the cathepsin B activities were completely inhibited by iodoacetamide and leupeptin, but phenylmethanesulfonyl fluoride and o-phenanthroline were ineffective. The molecular weight of the cathepsin B of the cellular origin and granuloma tissue was approximately 24,000 by gel filtration. The pI value of cathepsin B in the P-I and P-II fractions from granuloma tissue was 5.09 and 5.18, respectively. These results strongly suggest that two types of cathepsin B in granuloma tissue may be different in cellular origin and may be isozymes.

Anti-inflammatory activity of a proteolytic enzyme, Prozime-10.

Prozime-10 (P-10), a proteolytic enzyme extracted from cultured broth of Aspergillus melleus, was injected intravenously into rats in dosages of 1--10 mg/kg and was found to alleviate carrageenin edema. Thermal denaturation diminished not only the proteolytic activity but also the antiedematous effect. Carrageenin-induced granuloma pouches in rats were reduced by P-10 in a dose-dependent manner (1--5 mg/kg). With P=10 (5 mg/kg i.v.), adjuvant arthritis was inhibited by 62% in the primary phase and by 57% in the secondary phase. Serum concentration of immunoprecipitable P-10 reached 100 mg/l within 30 min after the intraduodenal administration of P-10 (200 mg/kg) and was concomitant with a 3-fold increase in the blood esterolytic activity. When P-10 was injected (5 mg/kg i.v.) into rats, the plasma corticosterone level increased to a maximum of 4 times the preinjection level. These observations indicate that: (a) P-10 can be absorbed from the gut in sufficient quantities to bring about anti-inflammatory activity, and (b) the anti-inflammatory activity of P-10 can be related, in part, to the ability of P-10 to affect the release of glucocorticoids.

In vivo metabolism of [1- 14C]arachidonic acid during different phases of granuloma development in the rat

The metabolism of [1- 14C]arachidonic acid was studied in vivo during the development of carrageenin-induced granuloma in the rat. For this purpose a double-cannulated teflon cylinder was implanted into the dorsal subdermal tissue of rats, and the cannulae were exteriorised through the skin at the scruff of the neck. The “chamber” allowed the injection of substances and collection of exudate at different stages of granuloma development. When [1- 14C]arachidonate was injected into the chamber one hour before recovery of the exudate at periods up to 12 days after initiating the inflammation, hydroxy fatty acids and prostaglandin E 2 appeared to be the main products formed. Only very small amounts of thromboxane B 2 and 6-ketoprostaglandin F 1α were detectable. These in vivo results are in marked contradiction to observations of other workers with granuloma tissue in vitro. The present findings are discussed in relation to the postulated negative-feedback function of E-type prostaglandins and a possible role of hydroxy fatty acids in inflammation.

Inhibition of thromboxane B 2 and 6-ketoprostaglandin F 1α formation by anti-inflammatory drugs in carrageenin-induced granuloma

Inhibition of thromboxane B 2 and 6-ketoprostaglandin F 1α formation by anti-inflammatory drugs in carrageenin-induced granuloma

Thromboxane B 2 transformed from arachidonic acid in carrageenin-induced granuloma

A main product (PI) transformed from arachidonic acid in carrageenin-induced granuloma in rats was structurally analysed. PI was converted to a more polar substance by the reduction with sodium borohydride. On the basis of the data on gas chromatography-mass spectrometry, its structure was identified with the hemiacetal derivative of 8-(1-hydroxy-3-oxopropyl)-9,12-heptadecadienoic acid (Thromboxane B2)

Modification of drug-metabolizing enzyme activity of rats in carrageenin-induced inflammation.

The activity of drug-metabolizing enzymes in hepatic microsomes of rats with inflammatory lesions was determined. In the Donryu-rats carrying carrageenin-induced granuloma, the cytochrome P-450 and b5 levels and the activity of aniline hydroxylase in hepatic microsomes were depressed markedly at day 6 after s. c. injection of carrageenin when the granuloma formation and exudate accumulation occur rapidly. However, at day 15 after carrageenin injection when the granuloma is in regression, the activity of these enzymes was decreased slightly but without significant differences.

A new prostaglandin transformed from arachidonic acid in carrageenin-induced granuloma

Summary A main product (PII) transformed from arachidonic acid in carrageenin-induced granuloma in rats was structurally analysed. PII was converted to a more polar substance by the reduction of sodium borohydride. According to the analysis of gas chromatography-mass spectrometry, its structure was speculated to be 9α,11α,15α-trihydroxy-6-oxo-prostenoic acid.

Effect of cyclic 3',5'-monophosphate on edema and granuloma induced by carrageenin

The anti-inflammatory activity of cyclic adenosine 3',5'-monophosphate (cAMP) alone or in combination with theophylline was examined in acute and chronic types of experimental inflammations in rats. cAMP suppressed carrageenin-induced edema by intraperitoneal, subcutaneous and oral administrations. In addition, cAMP inhibited the formation of carrageenin-induced granuloma. Systemic applications of cAMP also suppressed the leakage of circulating dye in rat skins induced by moderate thermal injury and by histamine and inorganic pyrophosphate, but they suppressed less effectively the leakage induced by exogenous bradykinin. Theophylline always augmented the suppressive effects of cAMP in these inflammations. A single intraperitoneal injection of cAMP alone or in combination with theophylline induced a slight, transient hyperglycemia and did not significantly alter the plasma level of corticoids. It seems unlikely that the anti-inflammatory action of cAMP is mediated solely by hyperglycemia or by increased production of corticosteroids.

Arylacethydroxamic Acids: a New Class of Potent Non-steroid Anti-inflammatory and Analgesic Substances

IN recent years much effort has been directed to the search for non-steroid anti-inflammatory substances which can be used in the therapy of rheumatic diseases and related conditions1. This communication reports the finding that a chemical family hitherto unexplored in this respect, namely, the arylacethydroxamic acids, includes several substances endowed with considerable anti-inflammatory and analgesic properties, and also a remarkably low degree of toxicity. A particularly interesting compound in this series was 4-butoxy-3-methyl-phenylacethydroxamic acid (I). In a comparative investigation with known antiphlogistic compounds, using the Benitz and Hall carrageenin-induced abscess test in rats2, compound (I), in exactly the same dose, proved only slightly less active (c. half) than phenylbutazone (l,2-diphenyl-3,5-diketo-4-butylpyrazolidine), definitely more active than ‘Ibufenac’ (p-isobutylphenylacetic acid), and considerably more so than aspirin. The activity shown by compound (I) at the high dosage of 1 g/kg was as intense as that of the maximum tolerated dose of phenylbutazone (400 mg/kg) and considerably more so than that (10 mg/kg) of ‘Indomethacin’ (1-p-chlorobenzoyl-5-methoxyindolyl-3-acetic acid), which is the most efficient non-steroid antiphlogistic substance known so far. Compound (I) was also strongly active in the carrageenin-induced granuloma test in rats, whereas phenylbutazone and aspirin are known to behave poorly in this type of assay3.

Morphological and chemical studies of collagen formation. II. Metabolic activity of collagen associated with subcellular fractions of guinea pig granulomata.

Electron micrographs of thin sections of nuclear, microsomal, and mitochondrial fractions obtained from a carrageenin-induced granuloma showed considerable contamination of the heavier by the lighter fractions. Striated collagen fibrils could be identified in the nuclei + debris fraction. Only a few striated fibrils occurred in the mitochondrial fraction; very fine filaments (diameter 50 A) could be seen in this fraction, but could not be distinguished with certainty from fibrillar material derived from broken nuclei. 35 per cent of the mitochondrial and 80 per cent of the microsomal collagen was extractable by 0.2 M NaCl and could be purified by the standard methods of solution and reprecipitation. The amino acid composition of these collagen fractions determined by ion exchange chromatography was within the range normally found for collagen and gelatin from other mammalian species, allowing for 10 to 20 per cent of some non-collagenous contaminant of the microsomal collagen. Hydroxyproline and proline were isolated by chromatography on paper from hydrolysates of the nuclear, mitochondrial, and microsomal collagen fractions, after incubation of tissue slices with L-(14)C-proline. The specific activities of the hydroxyproline from these collagens were in the approximate ratio 1:2:6, while that of bound hydroxyproline derived from the supernatant was only 1, indicating primary synthesis of collagen in the microsomes. Attempts to demonstrate incorporation of L-(14)C-proline into collagen or into free hydroxyproline in cell free systems were unsuccessful, nor was it possible to demonstrate non-specific incorporation of L-(14)C-valine into TCA-insoluble material by various combinations of subcellular fractions.