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The effect of the in vivo depletion of macrophage function on the plaque-forming cell (PFC) response to liposome-associated bovine serum albumin (LSM-BSA) in mice was investigated. Suppression of macrophage activities was accomplished by the intraperitoneal administration of carrageenan (CGN), a sulphated polygalactose which selectively inhibits macrophage function without interfering with the reactivity of T- and B-lymphocytes. Thus, control animals injected with CGN were found to give a significantly suppressed PFC response to sheep red blood cells, a macrophage-dependent antigen, but not to E. coli lipopolysaccharide, a macrophage-independent antigen. The pretreatment of experimental animals with CGN virtually abolished their antibody response to LSM-BSA in terms of the circulating anti-BSA antibody titers and the BSA-specific PFC response. These results provide evidence to substantiate an obligatory role of macrophages in inducing the humoral response to liposome-associated protein antigens.

An enzyme immunoassay was used to measure the levels of bradykinin (BK)-like immunoreactivity in inflammatory exudates provoked by calcium pyrophosphate (CaPP) or carrageenin. BK-like immunoreactivity increased from 4 to 18 ng/ml in exudates provoked by CaPP and collected from 30 min to 3 h after induction of pleurisy. BK levels decreased progressively (from 16 to 3 ng/ml) in the samples collected at 4 and 6 h. BK levels did not increase in exudates provoked by carrageenin. Control samples of exudates provoked by CaPP but not treated with peptidase inhibitors [orthophenanthroline (OPH) or a mixture of EDTA-captopril-thioglycolic acid (ECT)] as well as samples containing one or the other peptidase inhibitors dissolved in saline, were also negative. The present results indicate that an increased production of kinins is detectable only in exudates induced by CaPP and not in those provoked by carrageenin. Our data also suggest that the kinins are rapidly metabolized, both in vitro (in the exudate sample) and in vivo (in the rat pleural cavity).

Studies by a combination of vascular perfusion, histological and electron microscopic techniques show that subcutaneous injection of air into rats excites no acute inflammatory response, but leads to the formation of a pouch lined by avascular fibrous tissue. Existing subcutaneous blood vessels are displaced by the pouch but no new blood vessels are formed. By contrast injection of carrageenan with the air or into a preformed air pouch leads to a severe inflammatory reaction and the rapid development of a thick layer of granulation tissue. Either type of pouch is a useful system in which to study inflammation. However because the distribution and ultrastructure of small blood vessels in the pouch wall is quite different from the known characteristics of the microcirculation of synovial membrane, the air pouch is an unsuitable model for the study of injury to joints or experimental arthritis.

Collagen metabolism was studied in both, the developing and resorptive phases of carrageenin granuloma. The rate of collagen biosynthesis relative to the rate of total protein synthesis is almost twice as high during the deposit phase compared with the resorptive phase of the same process. Total collagenolytic activity appears to be approximately the same in both, deposit and resorption stages of the granuloma. Notwithstanding, the enzyme is fully active during resorption; whereas half of this activity is in a latent form during deposit. Collagenolytic activity increased remarkably when assayed at 30 mM CaCl2 as compared to 10 or 50 mM.

A single intraarticular injection of carrageenin into the rabbit knee joint initiates an inflammatory reaction in the synovial tissues. the exudate from the joint was able to degrade proteoglycan at pH 5.2 and pH 7.2. Further characterization of proteolytic enzymes in the inflamed synovial tissues showed the presence of cathepsin D, a neutral protease, and cathepsin B1. Maximum activities of two lysosomal enzymes, acid phosphatase and cathepsin D, were observed within 7 days of injection. Most of this activity was found to be associated with cells in the synovial fluid.

We have studied the ability of a quaternary antihistamine, thiazinamium, to inhibit the nociceptive sensitization that occurs early, during the first hour, following intraplantar injection of the polysaccharide carrageenin in the rat. Parallel studies were performed with an electrophysiological model (changes in responsiveness of ventro-basal thalamic cells driven by noxious stimulation of the paws), and a behavioural test (changes in threshold stimulus necessary to elicit vocalization by gradually increased pressure to the paws). When thiazinamium was given intravenously 10 min before carrageenin, no sensitization due to inflammation was found in either test. By contrast, when thiazinamium was administered 20 min after carrageenin, there was a clear sensitization in both tests that did not differ from that found in animals not treated with the antagonist. Paw oedema was also slightly decreased by pretreatment with thiazinamium. These results suggests that early inflammatory sensitization of peripheral nociceptors is mainly dependent on an initial release of histamine (and/or serotonin, since thiazinamium could also have some antiserotoninergic activity).

A localized inflammatory response was initiated by both single and repeated injections of carrageenin into femorotibial joints. Histologic changes were observed 24 hours after a single intraarticular injection, and an inhibition in the in vitro rate of proteoglycan synthesis was detected 72 hours after the injection. This inhibition was relieved in vitro by the addition of beta-D-xyloside, an exogenous initiator of glycosaminoglycan biosynthesis. Following repeated carrageenin injections, most cells appeared to be dead on histologic examination and no in vitro proteoglycan synthesis could be detected; nor could any stimulation be achieved by adding exyloside.

Among different carrageenins, kappa-carrageenans were found to be thrombogenic, whereas lambda-carrageenins were inactive in this respect, although the latter substances exerted a stronger edemogenic activity. Kappa-carrageenin (Sigma) was the most potent thrombogen. As the consequence of thrombosis tail infarction became visible some minutes after i.v. administration, but it was delayed for about 3 hours after the i.p. route and for about 6 hours after subplantar injection. Infarction frequency as well as extent of infarction was inhibited by heparin, cyproheptadine, phentolamine and dibenamine. Other substances like aspirin and dipyridamole showed no or only weak effects. Advantages of the carrageenin-induced thrombosis model in rats and mice are: (i) simple induction in small laboratory animals, (ii) easy observation and quantification all the time without killing the animals, and (iii) possible external testing of antithrombotic agents by applying substances on the tail.

An in vivo method is described showing how selective killing of macrophages in the peritoneal cavity by carrageenan affects Eimeria tenella infection in chickens. Killing of macrophages was demonstrated by the increasing loss of cytoplasmic contents under light microscopy, by the disappearance of mitochondria and endoplasmic reticulum ultrastructurally, and by the about 10%-per-hr loss of nonspecific esterase activity as uptake of carrageenan increased. Heterophils were apparently unaffected. Effect of such a selective killing on E. tenella infection seemed to be greatest when the carrageenan was injected intraperitoneally 24 hr before oocyst inoculation per os, but effect after the oocysts were inoculated was minimal or nonexistent. That chickens injected with carrageenan twice (once 24 hr before and once 48 hr after E. tenella inoculation) showed higher lesion scores with 15,000 oocysts than the controls showed with 60,000 oocysts further demonstrated the participation of macrophages in E. tenella infection.

Rats were fed either a basal purified diet, or that diet supplemented with 50 g/kg pectin or iota carrageenan for 50 days, and caecal microbial nitroreductase activity determined using p-nitrobenzoic acid, p-nitrophenol, 2,4-dinitrotoluene, nitrofurantoin and metronidazole as substrates. Both pectin and carrageenan increased the weight of caecal contents, and pectin also increased the number of bacteria per caecum. In contrast, carrageenan decreased the caecal bacterial population. Pectin significantly increased the rate of reduction of metronidazole and the rate of conversion of p-nitrobenzoic acid to p-aminobenzoic acid, while carrageenan significantly decreased the rate of reduction of every compound studied. The results demonstrate that microbial reduction of the nitro-group may be altered by diet, although the response found with one nitro-compound may differ from that seen with another substrate.

The suggestion that carrageenan suppressed the cutaneous manifestation of established delayed hypersensitivity (DH) by macrophage cytotoxicity could not be supported histopathologically. In a dose more than sufficient to suppress DH, carrageenan was undetectable by histochemical means in the macrophages of sple-n, lymph nodes, or bone marrow. It required concentrations of carrageenan threefold higher than the immunosuppressive dose to be detected in the above locations. Under no circumstances was macrophage cytotoxicity observed in these sites.

A number of direct and indirect immunologic consequences follow the exposure of the skin to ultraviolet radiation (UVR). One of the remote effects of UVR is the increased accumulation of lymphocytes within peripheral lymph nodes that drain irradiated skin sites. The present study was designed to evaluate the mechanisms responsible for this increased lymphocyte accumulation. Primary lymphocyte localization into draining regional lymph nodes was found to be markedly increased (up to 81%) by UVR exposure of the skin. The changes in lymphocyte localization correlated closely with increases in the number and density of lymphocyte-receptive endothelial structures (high endothelial venules, or HEV) within these regional lymph nodes. Because macrophages or their products are believed to maintain resting HEV expression and function, we assessed the role of these cells in the expansion of HEV due to UVR exposure. Our studies demonstrated that a peripheral injection of antigen-activated macrophages caused an increase in HEV expression within lymph nodes draining the injection site. Conversely, treatment of mice with silica or carrageenan to depress macrophage function produced both histologic and functional hyporesponsiveness of the microvascular endothelium to UVR-stimulated HEV expansion. These changes in endothelial cell responsiveness correlated with the absolute number of MAC-1+ cells present within the regional nodes of UVR-exposed mice. Our studies have demonstrated a striking similarity between lymph node microvascular changes in response to cutaneous UVR- and antigen-exposure. We therefore suggest that macrophage-mediated signals may initiate a general mechanism that increases lymphocyte recirculation through the regional lymph nodes in response to both antigenic and inflammatory challenge.

Tilorone suppressed inflammation induced by immune (direct passive Arthus reaction) as well as by non-immune agents (carrageenam-induced paw edema and abscess formation), if the compound is given 24 hr prior to the proinflammatory agonists. The non-immune anti-inflammatory effect is independent of the adrenals. A surprising findings was that total serum hemolytic complement is markedly elevated 24 hr after a single dose of tilorone.

When injected subcutaneously in the dorsum of neck in albino rats, carrageenan produced inflammatory swelling which reached peak after about 16 hr. The occurrence of the peak inflammatory swelling was delayed but not significantly reduced in severity by aspirin or indomethacin which were administered repeatedly. Phenylbutazone significantly reduced and dexamethasone almost completely inhibited it. In rat hind paw model, subplantar carrageenan injection produced peak inflammatory swelling after about 4 hr which was significantly reduced by all anti-inflammatory drugs mentioned above. It is interesting that an inflammagen when injected at different sites in the same species elicits responses which differ in the time course and drug responses.

PGI2, but not its metabolite 6oxoPGF1alpha, is equivalent in potency to PGE1 as a potentiator of carrageenan, histamine and bradykinin-induced rat paw oedemas. PGI2 must, therefore, be considered as a potential mediator of inflammatory processes.

The epidermis and corium of rat right hind paw were investigated both histologically and histometrically after a subplantar injection of aerosil, formalin, carrageenin or Freund's complete adjuvant. The thickness of the epidermis and corium was determined on 5 rats respectively at 12 different times between 0.5 and 480 h post-treatment. Paw swelling was measured plethysmometrically on living rats. The left paw of untreated rats as well as the saline-injected and the untreated left paw of carrageenin-treated and adjuvant-treated animals respectively served as controls. Results 1. After the injection of the 4 substances, an acute inflammation developed maximally 4 to 24 h post-treatment in the corium. The thickness of the corium increased simultaneously with paw swelling. 2. Epidermal hyperplasia set in after a lag-phase of 12 h following the injection of formalin, carrageenin and adjuvant. 3. After injection of aerosil a subcutaneous oedema developed but no epidermal hyperplasia occured. 4. The formation of granulation tissue and necroses in the corium was seen only in the adjuvant-treated group 4 to 20 days post-treatment. At the same time, epidermal hyperplasia reached an absolute maximum. 5. The disturbance of nutrition and the lack of oxygen in the inflamed corium is possibly responsible for the initiation and persistence of the hyperplasia.

In the rat paw prostacyclin was 5--10 times less potent than PGE2 in causing oedema, and 5 times less potent in potentiating carrageenin-induced oedema, which it did in a dose-related manner. Prostacyclin was 5 times more potent than PGE2 in producing hyperalgesia and as potent as PGE2 in restoring carrageenin-induced hyperalgesia. The effects on oedema were longer lasting than those on hyperalgesia. 6-oxo-PGF1alpha was 500 times less potent than PGE2 in causing oedema by itself and in potentiating carrageenin-induced oedema. It had no hyperalgesic activity in this test.

Iota-carrageenans are high molecular weight polygalactans commonly used in the food industry. Their immunomodulating effects were examined on the reaginic and IgG ovalbumin-specific responses after systemic or oral administration to Brown Norway rats. Intraperitoneal injection of ovalbumin with either iota-carrageenan or alum induced both a reaginic and IgG response. Reaginic antibodies were detected in the primary response with alum, but only in the secondary response with iota-carrageenan. Oral tolerance to ovalbumin was similarly induced whether the protein was given alone or admixed in solution with iota-carrageenan. These results confirmed the systemic adjuvant action of iota-carrageenan described earlier, and showed that this effect did not take place when orally administered.

Carrageenan, a high molecular weight sulphated polygalactan, is a potent inhibitor of immune responses mediated by macrophages. In the present study, spleen cells from rats orally dosed with 5 mg/kg or 50 mg/kg Seakem 9 carrageenan displayed a long-lasting depression of T lymphocyte mitogenesis as measured by [3H]-thymidine uptake in response to phytohaemagglutinin (PHA) or concanavalin A (Con A). Maximal suppression of splenic T cell proliferation occurred with the low dose (5 mg/kg) of orally administered carrageenan. Removal of adherent cells restored the PHA mitogenic response, suggesting a macrophage-mediated mechanism in suppression of lymphocyte activation. Rats which received 5 mg/kg carrageenan displayed impaired host resistance to Listeria monocytogenes as evidenced by increased numbers of Listeria in the peritoneal cavity 18 hr after i.p. inoculation. Supernatants from peritoneal exudate macrophages, as well as resident macrophages themselves obtained from carrageenan-fed rats, also suppressed PHA-induced spleen cell mitogenesis. These data support the hypothesis that low doses of orally administered carrageenan stimulate a population of macrophages to actively suppress T lymphocyte proliferation, while high doses abolish suppressor activity.

The subchronic and chronic toxicity of undegraded (native) carrageenan with respect to botanical source, and type of polymer chain (kappa, lambda, and iota) was investigated in rats for 13 to 39 weeks. The carrageenans were incorporated in the diet at 1 or 5% w/w. In all instances comparable levels of Alphacel were used. In the study designed to determine if there were any differences between various strains of rat, the Osborne-Mendel rat was used in addition to the Sprague-Dawley strain. Only the Sprague-Dawley strain was used in the other studies. Parameters utilized for detecting toxicity included mortality, body weights, fecal occult blood, hematologic indices, clinical chemistry, gross and microscopic histology, and evidence of carrageenan storage. Except for minor changes, no evidence was obtained for any direct effect of the carrageenans on the liver or the gastrointestinal tract.

Seventy-two women with proven detrusor instability completed a novel clinical trial of the new anticholinergic emepronium carrageenate (Cetiprin novum), which allowed for dose titration. The tolerated dose has been shown to correlate with the severity of symptoms. Statistically significant improvement was achieved in symptomatic and urodynamic parameters without serious side effects.

A single intraarticular injection of carrageenin into rabbit knee joints initiated a localized synovial inflammatory response. This response was accompanied by a 20% loss of proteoglycan from the articular cartilage within 24 hours and by a further 30-60% loss within 5-7 days. The chondrocytes replaced the lost proteoglycan within 42 days. More than two injections caused only a further small decrease in proteoglycan content; the cartilage was then unable to replace the lost proteoglycan. The absence of recovery coincided with the appearance of erosion of the cartilage surface.

Degraded carrageenan derived from the red seaweed Eucheuma spinosum was given to Sprague-Dawley rats through the diet, in drinking water or by stomach tube for up to 24 months. Carrageenan-induced squamous cell carcinomas, adenocarcinomas and adenomas in the colorectum were observed. Some rats had metastases to the regional lymph nodes of squamous cell carcinomas. These results show that degraded carrageenan is carcinogenic to the colorectum of the rat.

Carrageenan is sulfated polysaccharide which has been extensively used as emulsifier and thickening agent in the food industry, for its ability to induce acute inflammation in pharmacology and for its selectively toxic effect for macrophages in immunology. Carrageenan is a complex substance which displays various biological properties. The authors have shown the extent of these actions and reviewed the latest investigations on this subject.

A colitis-like state induced in Guinea Pigs fed degraded carrageenan orally. By means of a combined semimacroscopic and histologic technique the course of the disease was followed during 28 days. The changes were primarily seen and became most prominent in the caecum. The first lesions were observed following 24 hours of treatment as small rounded foci initially with degenerative changes and inflammation in the surface epithelium, later forming superficial focal ulcerations. Ulcerative changes gradually progressed during the experiment, forming linear and later large, geographical ulcerations. Topographically the ulcerative process was strongly related to the larger submucosal vessels. Nonulcerated parts of the mucosa were not changed until following 7-14 days of treatment. The mucosa became bulging, granulated and finally villus-like. Accumulation of macrophages was found under the surface epithelium after 7-17 days. Possible pathogenetic mechanisms are discussed, especially the development of the early lesions and the significance of the macrophages.

Rheumatoid arthritis commonly improves markedly during pregnancy, and it has been suggested that this reflects a decrease in immunological or inflammatory effector mechanisms. We elicited a delayed type hypersensitivity (DTH) response to Burro RBC, by injection of primed lymphocytes and antigen into murine footpads. The swelling induced in footpads of pregnant mice was not significantly different from that seen in controls, nor was the response to a non-immunologic inflammatory stimulus, carrageenan, decreased in pregnant mice. On the other hand, a defect in the priming or induction phase of a primary DTH response was impaired in pregnancy, but whether this is relevant to the pregnancy induced remission of rheumatoid arthritis remains to be demonstrated.

Silica (4 mg/i.v.) and Carragheenan (20 mg/i.p.) were shown to be capable of weakening hybrid resistance in the spleen whereas no improved growth was observed in the femoral bone marrow. Histological data did not indicate that the agents in the doses used caused significant macrophage mortality. It was concluded that the weakening effect of the agents was not due to death of the effector cells but possibly to a stimulation of the mononuclear phagocytic system in the spleen. Cyclophosphamide (250 mg/kg) induced a weakening of hybrid resistance which was observed both in the spleen and the femoral bone marrow. Experiments with the alkylating agent Busulphan and with Vinblastin showed that both agents were only capable of inducing a moderate stimulation of growth of the transplant in the spleen. A direct specific influence of cyclophosphamide on the microenvironment was proposed. Xenogeneic resistance was studied by transplantation of rat bone marrow to mice. Silica and cyclophosphamide were shown to be capable of inducing a weakening of the resistance in the spleen. The development of the graft in the femoral bone marrow did not appear to be affected by the administration of silica or cyclophosphamide.

1. Local injection of carrageenin in the rat's paw produced oedema and leakage of dye which had been administered previously by the intravenous route. A net dissociation between both parameters was observed: while oedema developed slowly, maximal intensity being attained after 4-5 h, dye-leakage was maximum after 1 hour.2. Anti-inflammatory drugs such as acetylsalicylic acid and indomethacin were effective in reducing oedema and dye-leakage when given before the injection of carrageenin, but much less effective when given 30 or 60 min after carrageenin. Hexadimethrine bromide was effective in reducing dye-leakage also when given 1 h after the injection of carrageenin.3. Combined administration of benadryl and methysergide, before the injection of carrageenin caused only a slight reduction in oedema and dye-leakage.4. When the paws were heated (55 degrees C for 30 s) as a noxious stimulus the dissociation between maximal oedema and maximal dye-leakage was not observed, both phenomena running parallel. Pre-treatment of the animals with indomethacin did not afford any protection.5. These results suggest that the inflammatory reaction to mild stimuli (carrageenin in our experiments) develops through different phases: initially the increased vascular permeability involves extravasation of plasma proteins and that phase is followed by an increased permeability mainly to water. Stronger stimuli (heating in our experiments) produce an overlapping of both phases, probably by inflicting severe damage to the vascular bed of the affected area.6. The anti-inflammatory drugs employed affected chiefly the initial phase of the response.