Secreted glucocorticoids regulate leukocyte-endothelial interactions in inflammation. A direct vital microscopic study.

Leukocytes come into intimate contact with the venular endothelium as they extravasate from blood to the interstitium during inflammation. In exteriorized tissues, the number of leukocytes rolling along the vessel wall was markedly increased in adrenalectomized and metyrapone-treated animals, relative to sham-operated and normal animals. During the development of an acute, local inflammatory response, rollers were numerically decreased and a stronger adhesion of the cells to the endothelium, with a concomitant migration into tissues, was observed. Adhesion and migration were much more marked in adrenalectomized and metyrapone-treated animals than in controls, suggesting that secreted glucocorticoids exert a suppressive effect on leukocyte-endothelial interactions. The increased number of rolling leukocytes in adrenalectomized and metyrapone-treated animals apparently resulted in more cells available to migrate into inflamed tissues. The effect appears to involve receptor occupancy and induction of gene expression because normal animals receiving the steroid antagonist RU 38 486, actinomycin D, or cycloheximide behaved as adrenalectomized or metyrapone-treated animals. Administration to adrenalectomized animals of a monoclonal antibody to the membrane glycoprotein complex CD18 did not affect the number of rolling cells, but dramatically reduced the number of adherent or migrated leukocytes. It is suggested that secreted glucocorticoids, in addition to an effect on rolling behavior of circulating leukocytes, might also modulate the activity of the glycoprotein complex CD18 on white blood cells. The ultimate consequence is a restrictive effect on cell emigration in inflammation.

Allergen-induced airway inflammation in rats. Role of insulin.

Clinical asthma appears to be less severe when diabetes mellitus is superimposed. To examine whether insulin influences the development of allergic reactions in the airway mucosa antigen challenge, normal and diabetic rats sensitized against ovalbumin (OA) were used. Compared with controls, animals rendered diabetic by the injection of alloxan presented markedly decreased cell yields from bronchoalveolar lavage after OA challenge. The impaired response was not related to antibody production because enhanced IgE antibody titers of the same magnitude were found in both control and diabetic animals. Similarly, the mechanism underlying the inhibited responses could not be ascribed to hyperglycemia or intracellular glucopenia, first, because correction of blood glucose levels through fasting did not restore the decreased response, and second, because administration of 2-deoxyglucose, which blocks glucose utilization, did not affect the bronchoalveolar reaction to OA challenge in normal animals. Reversal of the impaired responses was attained by treatment of diabetic animals with insulin. There is evidence that insulin exerts proinflammatory effects. We conclude that insulin might modulate the inflammatory component of asthmatic responses.

Hormonal control of inflammatory responses.

Almost any stage of inflammatory and immunological responses is affected by hormone actions. This provides the basis for the suggestion that hormones act as modulators of the host reaction against trauma and infection. Specific hormone receptors are detected in the reactive structures in inflamed areas and binding of hormone molecules to such receptors results in the generation of signals that influence cell functions relevant for the development of inflammatory responses. Diversity of hormonal functions accounts for recognized pro- and anti-inflammatory effects exerted by these substances. Most hormone systems are capable of influencing inflammatory events. Insulin and glucocorticoids, however, exert direct regulatory effects at concentrations usually found in plasma. Insulin is endowed with facilitatory actions on vascular reactivity to inflammatory mediators and inflammatory cell functions. Increased concentrations of circulating glucocorticoids at the early stages of inflammation results in downregulation of inflammatory responses. Oestrogens markedly reduce the response to injury in a variety of experimental models. Glucagon and thyroid hormones exert indirect anti-inflammatory effects mediated by the activity of the adrenal cortex. Accordingly, inflammation is not only merely a local response, but a hormone-controlled process.

Inhibition of neutrophil chemotaxis and chemokinesis associated with a plasma protein in aging rats: selective depression of cell responses mediated by complement-derived chemoattractants.

The influence of aging on neutrophil chemotaxis, chemokinesis, and superoxide production was investigated in rats. Animals of two age groups, 3 to 4 months and 20 to 21 months, were used. Equivalent neutrophil chemotactic responses to N-formyl-methionyl-leucyl-phenylalanine (fMLP), leukotriene B4 (LTB4), and bacterial lipopolysaccharide (LPS)-activated plasma were observed in both groups of animals, with cells suspended in Hanks' balanced salt solution (HBSS). However, cross-incubation studies in which cells from young adult rats were exposed to plasma from aged donors, then resuspended in HBSS for testing, showed marked changes in the ability of the cells to respond to the chemoattractants. The response to LPS-activated plasma was reduced, whereas responses to fMLP and LTB4 remained unaltered. Previous incubation of the cells with homologous plasma from young donors produced no effect. The inhibitory activity developing with advancing age affected not only chemotaxis but also random movement stimulated by LPS-activated plasma. The inhibitory activity of chemotaxis and chemokinesis in plasma of aged animals was heat labile (56 degrees C), vanished in the presence of a proteolytic enzyme like trypsin, and was maintained after dialysis with 12,000-Mr retention dialysis tubing. The material did not influence superoxide production by stimulated neutrophils. It is suggested that inhibition of neutrophil locomotion with advancing age is associated with a plasma protein capable of interacting with neutrophil receptors for complement-derived chemoattractants. The inhibitory substance might influence neutrophil responses to infection and inflammation in the elderly.

Direct vital microscopic study of defective leukocyte-endothelial interaction in diabetes mellitus.

The number of leukocytes rolling along the venular endothelium of the vascular network of the internal spermatic fascia was determined in nondiabetic control rats and diabetic rats with television microscopy. A marked decrease in the number of rolling cells was observed in animals rendered diabetic by the injection of alloxan 10, 30, or 180 days before relative to matching controls. Blood leukocyte counts, however, were equivalent in both control and diabetic rats. Under the influence of a local inflammatory stimulus, cells emerged into the perivascular tissue in control animals, and this was accompanied by a reduction in the number of rolling leukocytes. In diabetic rats, the number of rolling leukocytes remained unaltered, and only a few cells accumulated in the connective tissue adjacent to the vessel. Reversal of the defective leukocyte-endothelial interaction was attained by treatment of diabetic animals with insulin. Inhibitors of arachidonic acid metabolism were ineffective to improve leukocyte-endothelial interactions in diabetic animals. Control rats injected intravenously with lyophilized plasma constituents, obtained after dialysis of diabetic rat plasma with 12,000-Mr retention dialysis tubing, behaved as diabetic animals in that they exhibited a reduced number of leukocytes rolling along the venular endothelium. In contrast, material obtained from control rat plasma produced no effect. Heating of active samples for 1 h at 56 degrees C resulted in the complete loss of the inhibitory effect. We conclude that a substance or substances present in diabetic plasma induce a defective leukocyte-endothelial interaction that further impairs resistance to infection.

Inhibition of leukocyte chemotaxis by serum factor in diabetes mellitus: selective depression of cell responses mediated by complement-derived chemoattractants.

Rat neutrophil chemotactic responses to N-formyl-methionyl-leucyl-phenylalanine (FMLP), leukotriene (LT) B4, and lipopolysaccharide-activated serum (LPS-AS) were quantitatively assessed using the micropore filter system. Cells were suspended in either normal or diabetic rat serum for testing. Diabetic donor serum did not affect migration of neutrophils in a concentration gradient of the synthetic chemotactic agents. In contrast, the migratory responses to LPS-AS were significantly less than normal in this circumstance. Summation of effects was observed when FMLP and LPS-AS, or LTB4 and LPS-AS were simultaneously added to the test chamber, with cells suspended in normal serum. Suspended in diabetic rat serum neutrophils responded normally to the synthetic chemoattractants but the response to the activated serum was blocked. Cells previously incubated in the presence of diabetic donor serum then transferred to a culture medium for testing, presented reduced migratory responses to LPS-AS. Supramaximal, inhibitory concentrations of FMLP and LTB4, did not influence the response of neutrophils to LPS-AS. In vivo, suppression of cellular emigration to an inflamed area was observed from the early stages of the diabetic state. The inhibitory activity of chemotaxis in diabetes mellitus was previously reported to be associated with a protein factor in plasma of the animals. It is suggested that the inhibitory factor of chemotaxis in diabetes mellitus interacts with neutrophil receptors for complement-derived chemoattractants to induce blockade of cell-oriented locomotion either in vitro or in vivo.

Endothelin-1 induces potent constriction of lymphatic vessels in situ.

The response of lymph vessels, arterioles and venules in the exteriorized rat mesentery to endothelin-1, vasopressin and norepinephrine was examined with the aid of high-resolution television microscopy. On a molar basis, endothelin-1 was more potent than vasopressin to contract the three types of vessels. Norepinephrine, which could constrict blood microvessels, did not act on lymph vessels. Acetylcholine, sodium-nitroprusside and isoproterenol were ineffective to block the constrictive responses of lymph vessels to endothelin-1 and vasopressin. At the same concentrations, however, acetylcholine and sodium-nitroprusside antagonized the responses of arterioles and venules to endothelin-1 and norepinephrine, whereas the responses of blood microvessels to vasopressin remained unaffected. Isoproterenol, at doses capable of blocking the response of the arterioles and venules to norepinephrine, did not interfere with the constriction induced by endothelin-1 and vasopressin on these vessels. It is suggested that endothelin-1 might play a role in the regulation of lymphatic contractility apart from its vasoconstrictor activity on blood vessels.

Lymphatic transport of salicylates in dogs.

1. Pharmacokinetic parameters were determined for acetylsalicylic acid (ASA) and salicylic acid (SA) in plasma and lymph following the intravenous or oral administration of a water-soluble preparation of lysine-acetylsalicylic acid to dogs. 2. By both routes of administration, ASA but not SA, tended to be deposited in lymph, as indicated by the ratio between the area under the concentration-time curve constructed for the parent compound and its metabolite in lymph and plasma. 3. A reduced conversion of ASA to SA by esterases in lymph, and lymphatic absorption of ASA following the oral administration might be factors responsible for the accumulation of the compound in the lymphatic system. 4. It is suggested that the lymphatic system might serve as a temporary reservoir compartment for ASA.

Effect of endothelin-1 on arterioles and venules in vivo.

The vasoconstrictor effects of endothelin-1 (ET-1) and norepinephrine (NE) were compared in the exteriorized rat mesentery in situ. ET-1 was 1,000 times more potent than NE in constricting both arterioles and venules. Vasoconstrictions induced by ET-1 lasted much longer than those caused by the catecholamine. At nonrelaxant doses, acetylcholine and sodium nitroprusside antagonized the constrictive response of microvessels to ET-1 and NE. However, larger doses of acetylcholine were necessary to inhibit the response to ET-1 than that to NE.

Functional changes in the microcirculation of alloxan-induced diabetic rats.

1. The constrictor response of microvessels to norepinephrine and tyramine, and the dilator response to acetylcholine and papaverine was equivalent in normal and alloxan-induced diabetic rats. 2. Acetylcholine and phentolamine were equally effective as antagonists of the vasoconstrictor action of norepinephrine in both groups of animals. 3. The minimum effective doses of histamine and PAF-acether to produce maximum dilation of microvessels, or to antagonize the response to norepinephrine in normal rats had to be increased 20-fold to evoke equivalent effects in diabetic animals.

PAF-acether and endotoxin display similar effects on rat mesenteric microvessels: inhibition by specific antagonists.

The activity of platelet activating factor (PAF-acether) and the effects of specific antagonists BN 52021 and WEB 2086 on microvessels were studied. The mesentery of anaesthetized male Wistar rats was exteriorized and arranged for microscopic observation of the microcirculation in situ. Vessel diameters were measured with an image splitting micrometer adjusted to the phototube of the microscope. Images were displayed on the monitor screen of a closed-circuit television system. Topical application of PAF (10, 100 and 200 microM) enlarged the diameter of mesenteric arterioles and venules, which was antagonized by PAF antagonists administered i.v. (1 mg/kg) or topically (10 microM). Bolus injection (1 micrograms/kg) or perfusion (100 ng/kg/min) of PAF significantly decreased mean arterial blood pressure of the animals and slightly increased arteriolar diameters. These effects were blocked by the PAF antagonists. Endotoxin (10-20 mg/kg i.v.) significantly enlarged arteriolar diameters and produced a significant drop of arterial blood pressure. These effects were blocked by the PAF antagonists at doses equivalent to those used to antagonize the effects of PAF. The leukocyte activator fMLP, as well as lyso-PAF and PAF antagonists, were devoid of effects on the microcirculation when applied topically or injected i.v. We conclude that mesenteric microvessels contribute to the hypotensive effect of PAF and endotoxin and that PAF is released at the mesenteric area during endotoxic shock.

Platelet-activating factor (PAF-acether) and microcirculation: effect of Lyso-PAF and PAF-antagonists.

Topical application of a standard dose of noradrenaline (NA) to the anesthetized rat mesentery evoked a vasoconstrictor response the latency of which was increased in a dose-dependent way by previous addition of PAF-acether or histamine but not by Lyso-PAF. Lyso-PAF, however, blocked the antagonistic effect of PAF-acether to NA, the blockade being equivalent to that observed with PAF-antagonists (BN 52021 or WEB 2086) either injected iv, or applied topically. In contrast, the antagonistic action of histamine towards NA was not affected by Lyso-PAF or PAF-antagonists. Since platelet aggregation and leukocyte accumulation did not occur, PAF-acether appears to interact directly with microvessels to block vasoconstrictor stimuli, an effect antagonized by PAF-antagonists and the metabolite/precursor, Lyso-PAF.

Thyroid hormones and vascular reactivity: role of the endothelial cell.

Decreased maximal responses to noradrenaline, adrenaline and phenylephrine were observed in aortas isolated from rats pretreated with triiodothyronine and thyroxine for 8 days. In addition, a potent relaxant effect occurred when supramaximal concentrations of noradrenaline and adrenaline, but not of phenylephrine, were used. The relaxant effect was not observed in preparations in which the endothelium had been removed, and it was antagonized by propranolol added to the bathing fluid. Practolol was ineffective. In vivo, adrenaline and noradrenaline evoked pressor effects which were of shorter duration and smaller magnitude in hormone-treated animals than in the controls. Furthermore, a subsequent fall in blood pressure to below normal was observed, not only with adrenaline but also with noradrenaline, in animals receiving thyroid hormones. We conclude that when large amounts of thyroid hormones circulate in the body, large arteries are more prone to distention in the presence of increased quantities of released catecholamines, and resistance vessels are less responsive to their pressor effects due to a superimposing dilator effect. These changes might represent adaptative mechanisms in hyperthyroid states, and might depend on a greater sensitivity of endothelial beta 2-receptors to noradrenaline and adrenaline.

Involvement of lymphocytes in non-immune inflammation: dual effect of glucocorticoids.

Leucopenia induced by the administration of methotrexate reduced the volume of inflammatory exudate and the number of cells entering the pleural cavity during a 4-h carrageenin pleurisy when compared with that of non-leucopenic controls. The depressed response was partially but markedly restored when leucopenic animals were intravenously injected, immediately before the initiation of pleurisy, with spleen lymphocytes (or their products) obtained from normal, adrenal-demedullated or mock-operated rats. In contrast spleen lymphocytes (or their products) obtained from adrenalectomized rats or from rats receiving metyrapone, an inhibitor of adrenal glucocorticoid biosynthesis, were completely inactive. It is concluded that in physiologic concentrations glucocorticoids are essential for the production of lymphocyte-derived factors involved in the development of acute, non-immune inflammation. In pharmacologic concentrations, however, glucocorticoids suppress the release of such pro-inflammatory factors.

Endocrine disorders render rats hyporeactive to non-steroidal but not to steroidal anti-inflammatory drugs.

The effect of cortisol (10 and 20 mg kg-1 day-1, sc), indomethacin (2 and 4 mg kg-1 day-1, po) and piroxicam (10 and 20 mg kg-1 day-1, po) on the proliferative component of inflammation was investigated in normal, diabetic, adrenalectomized and diabetic-adrenalectomized rats using the cotton pellet test. Whereas cortisol was equally effective in preventing granulation tissue formation in all groups of animals, indomethacin and piroxicam were much less active in animals with hormonal dysfunctions. Indomethacin and piroxicam reduced thymus weight of normal and diabetic animals as much as cortisol. This was taken to be a strong indication of the effect of these non-steroidal drugs on the adrenal cortex leading to increased secretion of adrenal corticosteroids. We conclude that at least part of the anti-inflammatory effect of indomethacin and piroxicam, in the present experiments, can be ascribed to the release of endogenous corticosteroids. This would explain the decreased sensitivity of adrenalectomized animals to the non-steroidal anti-inflammatory drugs used. An additional component, however, seems to be necessary for the full expression of the anti-inflammatory effect of these drugs, since diabetic animals were also less responsive to them. When both components were absent, as in diabetic-adrenalectomized animals, indomethacin and piroxicam were practically devoid of an anti-inflammatory effect.

Vascular reactivity in diabetes mellitus: possible role of insulin on the endothelial cell.

The response to vasoactive agents of microvessels of the rat was tested in vivo by direct microscopic observation of the exteriorized mesentery and assessment of cutaneous vascular permeability changes with Evans blue. The constrictor response to a standard amount of noradrenaline in mesenteric microvessels was fully antagonized by acetylcholine in normal, diabetic, adrenalectomized and diabetic-adrenalectomized rats. In contrast, the minimum doses of histamine or bradykinin, effective in normal or adrenalectomized animals, had to be increased about 20 fold to be active in diabetic or diabetic-adrenalectomized animals. Topical application of insulin to mesenteric microvessels of diabetic animals, in amounts not causing any increase in serum insulin levels, improved or restored the capacity of the animals to respond to histamine or bradykinin, acting as antagonists of the vasoconstrictor response to noradrenaline. Topical insulin, however, was ineffective in normal animals given 2-deoxyglucose, the acute effects of which result from cellular glucopaenia unrelated to insulin deficiency. Vascular permeability responses to intracutaneous histamine or bradykinin were decreased in animals pretreated with 2-deoxyglucose as much as in diabetic animals. Pretreatment of normal animals with indomethacin produced no effect on the responses of these animals to histamine or bradykinin, tested as antagonists of noradrenaline on mesenteric microvessels, or as vascular permeability-increasing factors in the skin. Pretreatment of normal animals with chloroquine, mepacrine or dexamethasone had no effect on the reactivity of mesenteric microvessels to histamine and bradykinin, acting as antagonists to noradrenaline. 7 It is suggested that vasoactive substances, endowed with permeability-increasing properties, evoke relaxation of microvessels through an insulin-dependent action on endothelial cells, unrelated to the release of arachidonic acid metabolites. This action would lead to increased vascular permeability, with opening of interendothelial junctions, and temporary changes in composition of extravascular fluid, which in turn, would provide the basis for vasodilatation. Diabetes mellitus apparently impairs such responses as a result of the accompanying cellular glucopaenia. Adrenal corticosteroids are not involved in the impaired responses.

The inflammatory response of hyperthyroid and hypothyroid rats. Role of adrenocortical steroids.

The capacity to respond to inflammatory stimuli was tested in hyperthyroid and hypothyroid rats when thyroid defects, induced by hormone administration or thyroparathyroidectomy, respectively, were fully established. Whereas hyperthyroid rats presented consistently depressed inflammatory responses, hypothyroid rats responded in a normal fashion. Decreased reactions to intracutaneously injected histamine and serotonin, inhibited swelling reaction to carrageenin, injected into one of the hind paws, and depressed primary and secondary reactions to adjuvant (heat-killed M. tuberculosis), only occurred in the hyperthyroid group. In addition, only in this group of animals enlargement of the adrenal glands, reduced content of adrenal ascorbic acid, and decreased number of circulating eosinophils, which characterize a circumstance of adrenal cortical hyperactivity, were observed. A spontaneous reversal of the acute inflammatory response of hyperthyroid animals to carrageenin occurred 3-4 days after interruption of hormone administration, and this was coincidental with the return to normal of the previously enlarged adrenal glands. Similarly, specific inhibition of adrenal cortical steroid biosynthesis in hyperthyroid rats with aminoglutethimide, restored the previously depressed response to carrageenin, without interference with the increased levels of seric thyroxin, thus suggesting that the inhibitory effects of thyroid hormones on inflammatory responses are likely to be indirect. It is concluded that an excess of circulating thyroid hormones, but not their deficiency, can impair the development of inflammatory reactions, and that this effect, at least partially, depends on an increased secretion of adrenal corticosteroids.