Control of mast-cell 5-lipoxygenase.

Leukotrienes (LTs) from mast cells make important contributions to early events in inflammation. Therefore, the control of their 5-lipoxygenase was studied. We observed that culture conditions can significantly alter LT synthesis and intracellular 5-lipoxygenase levels. Challenge of mast cells with calcium ionophore A23187 and immunoglobin E (IgE)/antigen had different effects. The calcium ionophore caused continuous LT formation that was accompanied by the translocation of the enzyme to membranes as well as substantial loss of activity. In contrast, with receptor-mediated stimulation, IgE/antigen, enzyme inactivation was insignificant, and membrane binding and LT synthesis were transient. Addition of a calcium chelator stopped ionophore-induced LT production and inactivation of the 5-lipoxygenase and reversed its membrane association. Therefore, receptor-mediated activation of 5-lipoxygenase differs from that by calcium ionophore. The data also indicate that calcium regulates the membrane binding and dissociation of the 5-lipoxygenase. However, with excessively high calcium concentrations activity is lost.

Interleukin 8 and mast cell-generated tumor necrosis factor-alpha in neutrophil recruitment.

In the reverse passive Arthus reaction in mouse skin and immune injury of mouse dermal basement membrane, neutrophil (PMN) infiltration in mast cell deficient WBB6F1-W/Wv (W/Wv) mice was only 40% of that in WBB6F1-(+)/+ (+/+) mice that had a normal mast cell repertoire. An anti-tumor necrosis factor-alpha (TNF-alpha) monoclonal antibody (mAb) decreased PMN infiltration by 35-80% in +/+ but not W/Wv mice. In addition, an anti-human interleukin-8 (IL-8) MAb, DM/C7, inhibited PMN infiltration of the skin induced by either intradermal administration of recombinant human IL-1 beta or immune complex deposition. In both models of immune complex injury, DM/C7 reduced PMN infiltration by 40-60% in +/+ mice but not W/Wv mice. PMN infiltration and the sensitivity of this infiltration to anti-TNF-alpha or DM/C7 MAb in W/Wv mice whose mast cell population had been restored was indistinguishable from the influx observed in +/+ mice. These data suggest that TNF-alpha, IL-8, and mast cells play a fundamental role in PMN recruitment following immune complex injury.

Mast cell degranulation induced by type 1 fimbriated Escherichia coli in mice.

The strategic location of mast cells at the host-environment interface and their ability to release potent mediators of inflammation have suggested that these cells may play a pivotal role in host defense against bacterial infection. The ability of the opportunistic pathogen, Escherichia coli, to induce degranulation of mast cells obtained from the mouse peritoneum was investigated. We determined that unlike a mutant derivative deficient in the FimH subunit of the fimbriae or nonfimbriated E. coli, type 1 fimbriated E. coli induced mast cell degranulation in vitro. The magnitude of mast cell degranulation was directly proportional to the number of adherent bacteria on the cell surface in the initial period of the interaction. Using a mouse model of bacterial peritonitis, we demonstrated mast cell degranulation and histamine release by type 1 fimbriated bacteria in vivo. Furthermore, beads coated with FimH but not with FimA, the major subunit of type 1 fimbriae, evoked mast cell release of histamine in vivo in amounts comparable to that elicited by type 1 fimbriated E. coli. These studies reveal that mast cells can be degranulated by interaction with type 1 fimbriated E. coli and that FimH, the mannose-binding component of the fimbriae, is a potent mast cell stimulant.

Mast cell phagocytosis of FimH-expressing enterobacteria.

Most studies of mast cells have been directed at their role in the pathophysiology of IgE-mediated allergic reactions with little recognition of their participation in bacterial infections. We report that mast cells can specifically bind FimH, a mannose-binding subunit on type 1 fimbriae expressed by Escherichia coli and other enterobacteria. This interaction triggers mast cell phagocytosis and killing of the bacteria within vacuoles and through the release of superoxide anions. Also, in view of the fact that mast cells have the capacity to release inflammatory mediators and are particularly abundant in the skin, mucosal surfaces, and around blood vessels, we suggest that these cells play an important role in host defense against microbial infection.

Neutrophil elicitation in the reverse passive Arthus reaction. Complement-dependent and -independent mast cell involvement.

Previous investigations in our laboratory have shown that mast cells play a significant role in the initiation of immune complex-mediated inflammation. Histamine, leukotrienes, and TNF released from mast cells are important mediators of early inflammatory processes. In the peritoneal reverse passive Arthus reaction, we observed a biphasic release of TNF. Mast cells were responsible for the first peak. The complement system is also known to be central to the expression of antibody-induced immune injury. Therefore, in this study, we investigated the significance of activated complement in regulating mast cell stimulation and neutrophil recruitment in the peritoneal reverse passive Arthus reaction. Mast cell degranulation and the release of TNF during the initiation of inflammation were blocked by decomplementation and C5 deficiency. Mast cell degranulation later in the reaction was complement-independent. Therefore, mast cells were activated in vivo in antibody-mediated injury by two different mechanisms, early in the reaction by complement and later by an unknown stimulus. Both mast cells and intact complement were also required for the full expression of neutrophil influx and release of TNF in the later phase. In fact, activated complement and mast cell mediators seemed to be the only factors necessary for the initiation of neutrophil recruitment. The findings significantly contribute to the understanding of the mechanisms involved in the induction of inflammatory processes in immune complex-mediated injury.

Effect of culture conditions on mast cell eicosanoid synthesis.

When mast cells are grown in culture from mouse bone marrow cells, some cells are in suspension and some attach. The cells in suspension released leukotrienes and thromboxane when stimulated. Removal from the attached cells increased eicosanoid formation in the suspended cells two- to six-fold. The upregulation of arachidonic acid metabolism seemed to be due to an increase in phospholipase activity. The attached cells resembled macrophages in morphology, but stained positively for mast cells and produced PGD2 an arachidonic acid metabolite which is characteristic for mast cells. The attached cells did not synthesize eicosanoids when stimulated with IgE/antigen or A23187. Addition of arachidonic acid caused cyclooxygenase product formation but not leukotrienes. No 5-lipoxygenase was detected by immunoblot analysis. Lack of eicosanoid synthesis was, therefore, due to the loss of 5-lipoxygenase and phospholipase activity, while the cyclooxygenase was preserved. These experiments demonstrate that attachment of mast cells to plastic can significantly alter their eicosanoid metabolism.

Reversible translocation of 5-lipoxygenase in mast cells upon IgE/antigen stimulation.

The 5-lipoxygenase catalyzes the first two steps in the metabolism of arachidonic acid to leukotrienes. It has been shown that the calcium influx into leukocytes following stimulation by A23187 activates the enzyme and causes its translocation to the membrane. Leukotrienes are formed, and then the enzyme seems to be irreversibly inactivated. In the present investigation we have compared the effect of receptor-mediated mast cell activation (IgE/antigen) and A23187 on 5-lipoxygenase activity and translocation to the membrane. In contrast to the ionophore, IgE/antigen, which initiated the formation of similar amounts of leukotrienes as A23187, caused minor inactivation of the enzyme. Immunoblot analysis demonstrated that after antigen the membrane association of the 5-lipoxygenase was reversible, while with A23187 the translocation continued during the time of observation (60 min). Addition of a calcium chelator after ionophore challenge, prevented further inactivation of the enzyme and reversed its membrane binding. The data suggest that the continuous influx of calcium with A23187 is responsible for the extensive inactivation of the 5-lipoxygenase. In contrast, during receptor-mediated stimulation the transient increase in intracellular calcium seems to conserve the enzyme activity.

Neutrophil recruitment by tumor necrosis factor from mast cells in immune complex peritonitis.

During generalized immune complex-induced inflammation of the peritoneal cavity, two peaks of tumor necrosis factor (TNF) were observed in the peritoneal exudate of normal mice. In mast cell-deficient mice, the first peak was undetected, and the second peak of TNF and neutrophil influx were significantly reduced. Antibody to TNF significantly inhibited neutrophil infiltration in normal but not in mast cell-deficient mice. Mast cell repletion of the latter normalized TNF, neutrophil mobilization, and the effect of the antibody to TNF. Thus, in vivo, mast cells produce the TNF that augments neutrophil emigration.

Role of mast cells in plasma permeation due to immune injury of the skin basement membrane.

Immune injury of the basement membrane occurs in various human diseases. In the present study, an antibody specific for the basement membrane of mouse skin was injected i.d. into mast cell-deficient WBB6F1-W/Wv mice and their congenic controls, WBB6F1-(+/+). Vascular permeability changes, oedema and fibrin deposition were assessed. Plasma permeation, evaluated by dye exudation, was time and dose dependent in both groups of animals, but significantly less in WBB6F1-W/Wv than in normal mice. At 30 min, the time of maximum in congenic controls, extravasation of the dye was 60% less in mast cell-deficient than in WBB6F1-(+/+) mice. Pyrilamine decreased exudation by 40% in normal but not in WBB6F1-W/Wv mice, indicating that the mast cell mediator histamine contributes to the increase in vascular permeability. Mast cell deficiency also markedly reduced fibrin deposition as assessed by direct immunostaining. Oedema, measured as skin thickness, was 60% less in WBB6F1-W/Wv mice than in their congenic controls. A 5-lipoxygenase blocker inhibited plasma exudation and oedema in normal but not in WBB6F1-W/Wv mice. This indicates that leukotrienes are involved in these processes and that mast cells are important for their production. Local mast cell reconstitution restored dye extravasation and oedema to normal levels as well as the effect of the 5-lipoxygenase inhibitor. These findings show that mast cells and their mediators participate in these inflammatory processes which were initiated by the deposition of IgG on the skin basement membrane.

Mast cells enhance the antibody-mediated injury of skin basement membrane in mice.

Immunologic basement membrane injury occurs in certain human diseases. We investigated the role of mast cells in the initiation of inflammation induced by selective deposition of antibody on the basement membrane in the skin. Intradermal injection of the antibody into mast cell-deficient WBB6F1-W/Wv mice and their congenic controls, WBB6F1-+/+, caused C (C3) deposition and tissue damage preferentially at the dermo-epidermal junction (basement membrane). Damage occurred earlier and was more extensive in normal than in WBB6F1-W/Wv mice. Hemorrhage in WBB6F1-W/Wv was reduced by 50%. In both groups of mice, a dose- and time-dependent neutrophil infiltration reached maximum at 8 h. At the peak, neutrophil accumulation in WBB6F1-W/Wv was only 50% of that in normal mice. Mast cell reconstitution of WBB6F1-W/Wv mice normalized the inflammatory response. Pretreatment with a 5-lipoxygenase inhibitor, A-63162, reduced neutrophil infiltration by 60% in normal but not in WBB6F1-W/Wv mice. Mast cell repletion restored the effect of A-63162. The results indicate that mast cells are important for the initiation of inflammation induced by the deposition of antibody on the basement membrane and the production of leukotrienes participating in neutrophil elicitation.

Mast cells contribute to fibrin deposition in reverse passive Arthus reaction in mouse skin.

The activation of the clotting system is an important process during inflammation to contain the injury and initiate tissue repair. In the present study, we investigated the effect of mast cells on fibrin deposition in reverse passive Arthus reaction in mast cell-deficient WBB6F1-W/Wv(W/Wv) and control WBB6F1-(+)/+(+/+) mice, that were given 125I-labeled fibrogen intravenousty. An antibody dose-dependent increase in radioactivity was observed in the challenged skin sites. Sequential water and urea extractions characterized the radioiodinated fibrinogen derivatives present in the tissue. The radioactivity found in the various fractions of the stimulated samples from +/+ was 2-10-fold higher than that in specimens from W/Wv mice. The greatest difference was observed in the urea-insoluble pellet (cross-linked fibrin and its early degradation products). Reconstitution of W/Wv mice with mast cells augmented the response to levels similar to those in +/+ mice. Pretreatment with the antihistamine pyrilamine blocked the accumulation of 125I-labeled fibrinogen and its derivatives by approximately 70% in +/+ but not in W/Wv mice. Inhibition of leukotriene synthesis by A-63162 markedly decreased the accumulation of iodinated fibrinogen in both +/+ and W/Wv mice. The data suggest that mast cells and their vasoactive mediator histamine contribute to the exudation of clotting factors, which results in fibrin deposition and that mast cells also enhance fibrin cross-linkage.

Mast cell mediators regulate vascular permeability changes in Arthus reaction.

Plasma exudation characterizes the early phase of acute inflammation. The possible role of mast cells and their mediators in this event in immune complex-induced injury was studied. Dye exudation was assessed from 5 min to 2 hr after initiating reverse passive Arthus reaction in mast cell-deficient mice, WBB6F1-W/Wv (W/Wv), and their normal congenic controls, WBB6F1-+/+ (+/+). The response to antibody (10, 30 and 100 micrograms/site, i.d.) was dose- and time-dependent in both groups of mice. At the lower doses of antibody, 10 and 30 micrograms/site, exudation was significantly less (30% and 40%, respectively) in W/Wv as compared to +/+ mice between 15 to 45 min. With 100 micrograms of antibody/site, significant differences between W/Wv and +/+ mice were noted only at 15 and 30 min. The deficit in permeability changes in W/Wv mice was reversed by local mast cell reconstitution. In +/+ mice, pyrilamine and methysergide pretreatment reduced vascular permeability to the same extent by 70, 60 and 35% when stimulated for 30 min with 10, 30 and 100 micrograms of antibody/site, respectively. An equivalent inhibition was observed with the 5-lipoxygenase inhibitor A-63162. None of the inhibitors decreased plasma permeation in W/Wv mice. These results indicate that the mast cell mediators histamine and serotonin regulate vascular permeability early during an immune complex-mediated inflammation. The data also suggest the involvement of leukotrienes and the importance of mast cells in their synthesis. The profile of inhibition in +/+ mice agrees well with the difference in exudation observed between normal and mast cell-deficient mice.

Mast cell granules within endothelial cells: a possible signal in the inflammatory process?

Mast cell-endothelial cell interactions have long been suspected to be important in inflammation. The detail of the pathways of communication have yet to be elucidated. In this report we describe, for the first time, mast cell granules within endothelial cells of colonic capillaries in a patient with florid colitis, as well as rats with experimentally induced mast cell degranulation. We postulate that this phenomenon may occur more generally during immunologically mediated inflammatory cell degranulation, and may be one mechanism of communication to endothelium.

Leukotriene formation by mouse connective tissue mast cells.

Stimulation of peritoneal cells from BALB/c, CBA/J or WBB6F1(-)+/+ mice with IgE/antigen caused the release of mast cell granules and leukotriene C4. No leukotriene formation was observed with peritoneal cells from mast cell-deficient WBB6F1-W/Wv mice. Mast cells (greater than 98% purity), separated on metrizamide gradients, did not synthesize detectable amounts of leukotriene C4 when challenged immediately after purification. Co-culture of the mast cells with 3T3 fibroblasts restored the capability of the mast cells to produce leukotrienes. Addition of IL-3 during culture enhanced the synthesis of this eicosanoid. The 5-lipoxygenase inhibitor A-63162 blocked the leukotriene formation. Western blot analysis confirmed the presence of 5-lipoxygenase in connective tissue mast cells. These experiments demonstrate that mouse peritoneal (connective tissue) mast cells can produce significant amounts of leukotrienes.

Mast cells are critical for the production of leukotrienes responsible for neutrophil recruitment in immune complex-induced peritonitis in mice.

Mast cells are secretory cells strategically located in the vicinity of blood vessels where they can readily initiate and modulate various inflammatory processes, including plasma exudation and leukocyte infiltration. We have previously shown that 50% of the neutrophil influx during immune complex peritonitis in mice is due to mast cells. Eicosanoids are important mediators of various inflammatory processes including neutrophil infiltration. The possibility that mast cells are essential for the production of leukotrienes (LT) involved in the elicitation of neutrophils in immune complex peritonitis was investigated in mast cell-deficient, WBB6F1-W/WV, and normal, WBB6F(1-)+/+, mice. The time course and amounts of immunoreactive PGE2, 6-keto-PGF1 alpha, and TX3B2 released into the peritoneal exudates were similar in both sets of mice. LTB4 and LTC4 levels, however, were twofold higher in +/+ than in W/WV mice 2 h after stimulation. HPLC analysis of the peritoneal exudate confirmed the presence of leukotrienes. The 5-lipoxygenase inhibitor A-63162 blocked leukotriene production in a dose-dependent manner in both sets of mice. However, this compound caused a significant reduction (60%) of neutrophil infiltration only in WBB6F(1-)+/+ but not in the mast cell-deficient mice. Mast cell reconstitution of WBB6F1-W/WV mice restored the effect of A-63162 on PMN recruitment. These data suggest that mast cells in the vicinity of blood vessels are important for the synthesis of leukotrienes responsible for PMN recruitment.

Augmentation of reverse arthus reaction by mast cells in mice.

Immune complex-induced injury is an important pathogenic factor in antibody-mediated nephritis, systemic lupus erythematosus, rheumatoid arthritis, and other diseases. In this study we investigated the role mast cells in immune complex-mediated injury in mouse skin. Reverse Arthus reaction was induced in mast cell-deficient WBB6F1-W/Wv mice and their congenic controls (WBB6F1(-)+/+). Serial skin sections were evaluated for neutrophil infiltration, edema, and hemorrhage. In WBB6F1-W/Wv mice the neutrophil influx was only 40% and edema 60% of that in congenic controls. Hemorrhage was also significantly reduced in the mast cell-deficient mice. After mast cell reconstitution, the magnitude of the reaction in WBB6F1-W/Wv was equivalent to that in WBB6F1(-)+/+ mice. Mast cell release in reverse Arthus reaction was evaluated by measuring fluorescence intensity after avidin-FITC staining of mast cell granules. There was a 70% decrease in fluorescence intensity. The 5-lipoxygenase inhibitor A-63162 significantly decreased neutrophil accumulation (40%), edema (60%), and hemorrhage in WBB6F1(-)+/+, but not in mast cell-deficient mice. Mast cell reconstitution of WBB6F1-W/Wv mice restored the effect of A-63162. The results indicate that mast cells and their mediators, including leukotrienes, make an important contribution to reverse Arthus reaction.

The sensitized liver represents a rich source of endogenous leukotrienes.

The ability of livers to produce endogenous leukotrienes after immunological stimulation was tested with organs from rats and guinea pigs. Passive sensitization of rats in vivo with monoclonal murine antidinitrophenol-IgE before antigen challenge in the isolated perfused liver system elicited a rapid hepatic production and biliary excretion of leukotrienes as judged by radioimmunoassay after separation of individual leukotrienes by high-performance liquid chromatography. Within 10 min after antigen infusion, mainly leukotriene C4, but also leukotriene D4 and N-acetyl-leukotriene E4, appeared in the bile. The biliary excretion rate of antigen-induced cysteinyl leukotrienes rose from less than 2 pmol.min-1.(kg body mass)-1 before challenge to about 30 pmol.min-1.(kg body mass)-1 for 20 min before it declined toward prechallenge level. Quantitatively similar hepatic production of cysteinyl leukotrienes was elicited in isolated perfused guinea pig livers challenged with ovalbumin after active sensitization of the animals with ovalbumin plus Al(OH)3. To exclude extrahepatic contributions to the observed leukotriene production, both passive sensitization with anti-dinitrophenol-IgE and subsequent antigen challenge were performed on isolated rat livers perfused with blood-free medium. Such exclusively hepatic sensitization and challenge also resulted in massive production of leukotrienes. The biliary excretion rate of cysteinyl leukotrienes amounted to approximately 20 pmol.min-1.(kg body mass)-1 during the 10 to 20 min period after antigen challenge as compared with less than 1 pmol.min-1.(kg body mass)-1 before challenge.(ABSTRACT TRUNCATED AT 250 WORDS)

Modulation of mast cell eicosanoid synthesis by attachment.

The experiments discussed above indicate that a change in phenotype occurs when cultured mast cells attach. This modification in phenotype is associated with a downregulation of their own eicosanoid production and that of cocultured mast cells in suspension. The eicosanoid production of the floating mast cells is markedly elevated after removal from attached cells. This enhancement appears to be due to an upregulation of phospholipase activity. Experiments with actinomycin D and cycloheximide indicate that new protein synthesis is necessary for the increase to occur. This suggests that either new enzyme and/or peptides modulating phospholipase are generated.

The importance of mast cells for the neutrophil influx in immune complex-induced peritonitis in mice.

The role of mast cells in polymorphonuclear leukocyte (PMN) influx in Ag-antibody complex-induced peritonitis was evaluated in mast cell-deficient WBB6F1-W/Wv (W/Wv) mice and their normal littermates, WBB6F1-+/+ (+/+). Peritoneal cell influx was evaluated after i.p. injection of preformed immune complexes. The first significant elevation in the PMN count over PBS-treated controls in +/+ mice was observed 2 h after stimulation. During the period of maximum leukocyte concentrations (6 to 10 h), the increase in total cell count was 5-fold and in PMN 25-fold. In W/Wv mice the PMN influx started 2 h later than in the +/+ mice, and the maximum response (8 to 10 h) was only 50% of that in controls. Reconstitution of mast cells in W/Wv mice for 2 wk or more restored the PMN response to immune complexes. Mast cell release due to AG-antibody complexes was evaluated by measuring fluorescence intensity after berberine sulfate staining for heparin in mast cells from unstimulated as well as stimulated +/+ mice. There was a significant decrease in fluorescence intensity as early as 15 min after stimulation. By 30 min the fluorescence intensity had declined by 65%. This indicates extensive mast cell release that started before PMN mobilization. These experiments demonstrate that mast cells make a significant contribution to immune complex-induced inflammation.