J. Brom

Studies on the Mechanisms of Granulocyte Dysfunctions in Severely Burned Patients—evidence for Altered Leukotriene Generation

The leukotriene generation (LTB4, 20-OH-LTB4, 20-COOH-LTB4) from PMNs of severely burned patients (n = 6) was studied by reversed-phase HPLC. Granulocytes from all patients showed a decrease in leukotriene generation which only returned to normal levels when the patients recovered from their injuries. The leukotriene generation induced by different stimuli, i.e., the Ca++-ionophore A23187 (7.3 microM) or opsonized zymosan (2 mg) in the presence of exogenous arachidonic acid (60 microM) showed similar stimulation profiles. The cellular differentiation of the respective granulocyte fractions revealed that the decreased leukotriene generation was accompanied by the occurrence of immature granulocytes in the peripheral blood. Furthermore, the studies in the presence of exogenous arachidonic acid showed that the defect in leukotriene generation from granulocytes of surviving patients was due to the availability of metabolizable substrate (i.e., free arachidonic acid). Granulocytes from one nonsurviving patient showed in addition a defect in the metabolic ability of arachidonic acid to generate the respective leukotrienes. The generation of reactive oxygen species did not correlate with the observed alterations in the formation of the leukotrienes.

Metabolism of leukotriene B4 by polymorphonuclear granulocytes of severely burned patients

Leukotriene B4 release from polymorphonuclear granulocytes of severely burned patients was reduced as compared to healthy donor cells. This decrease is due to an enhanced conversion of LTB4 into the 20-hydroxy- and 20-carboxy-metabolites and further to a decreased LTB4-synthesis. In addition, studies on the exogenous LTB4-conversion revealed an unidentified compound which was derived from LTB4. Our data suggest a modulation of the enzymatic activities involved in omega-oxidation of LTB4 (isoenzymes of cytochrome P-450).

Cilastatin (MK 0791) is a potent and specific inhibitor of the renal leukotriene D4-dipeptidase

The metabolism of leukotriene D4 to leukotriene E4 by a dipeptidase of kidney tissue is strongly inhibited by cilastatin (MK 0791) a known renal dehydropeptidase-I inhibitor. The comparison with similar enzyme activities from other tissues (liver, lung, serum, polymorphonuclear granulocytes) revealed a high specificity of cilastatin for the kidney enzyme which was found to be associated with the microsomal fraction. The lowest detectable inhibitory concentration of cilastatin within renal tissue was 8 X 10(-8)M.

Functional characteristics of leukotriene C4- and D4 -metabolizing enzymes (γ-glutamyl transpeptidase, dipeptidase) within human plasma

Several properties of the leukotriene C4- and leukotriene D4-metabolizing enzymes within human plasma were studied after fractionation of the plasma proteins using ammonium sulfate precipitation. Leukotriene D4-metabolizing enzymes were widely distributed among the fractions obtained. They showed different pH optima (pH 6.5, pH 7.0 and pH greater than or equal to 8.5) and revealed a different degree of thermal stability. The results indicate the presence of more than one enzyme in plasma which interacts with leukotriene D4. EDTA and L-cysteine inhibited the metabolism of leukotriene D4. Two leukotriene C4-metabolizing activities (gamma-glutamyl transpeptidases) differing in their molecular weights were detected after gel filtration. Their molecular weights were estimated to be Mr greater than or equal to 150 000 and Mr between 55 000 and 100 000.

Characterization of leukotriene B4-omega-hydroxylase activity within human polymorphonuclear granulocytes.

Human polymorphonuclear granulocytes (PMN) metabolize exogenous [3H]leukotriene B4 (LTB4) into 20‐hydroxy‐ and 20‐carboxy‐[3H]LTB4. The conversion was enhanced at acidic pH values (pH 6.0–7.0). Sonication of purified PMN and subcellular fractionation by differential centrifugation showed that major LTB4‐hydroxylase activity was associated with the microsomal fraction (105,000 g pellet). In contrast to intact cells. LTB4‐hydroxylase activity within the microsomal fraction revealed optimal activity at neutral pH and was inhibited by a wide range of divalent cations. There was a strict requirement for the presence of suitable electron donors such as NADPH, Heterocyclic nitrogenous bases, such as imidazole and pyridine, inhibited the LTB4, conversion induced by intact PMN as well as by their microsomes. These observations combined with the spectrophotometric analysis (carbon monoxide dithionite‐reduced difference spectrum) supported the assumption that LTB4‐hydroxylase resembled a cytochrome P‐450 enzyme. The LTB4‐hydroxylase within human PMN was not identical with the cytochrome P‐450 of rat liver; hepatic microsomes only showed minute conversion of LTB4.

Decreased expression of leukotriene B4 receptor sites on polymorphonuclear granulocytes of severely burned patients

Polymorphonuclear granulocytes were isolated from patients with burn injury and the specific binding of (3H)leukotriene B4 was assessed. We observed a decreased receptor expression as compared to healthy donor cells, which may be the result of receptor downregulation as a consequence of cellular preactivation. In addition, leukotriene B4-synthesis was also reduced and differential cell counts demonstrated a shift from segmented neutrophils to immature cells. In survivors the values returned to normal parameters whereas nonsurvivors who succumbed in the course of generalized sepsis showed depressed cellular functions up to their death.

Altered arachidonic acid metabolism in granulocytes of polytraumatized patients

The generation and metabolism of leukotrienes (LTs) from polymorphonuclear granulocytes of four polytraumatic patients on stimulation with the Ca-Ionophore A 23187 were studied by high performance liquid chromatography. In contrast to healthy donors the concentration of LTB4 within the supernatant of the stimulated granulocytes from these patients is reduced. The ratio of LTB4 versus the combined amounts of the biologically inactive 6-trans and 12-epi-6-trans-isomers is significantly decreased. In one patient who suffered from an Adult Respiratory Distress Syndrome (ARDS) a pronounced enhancement of LTC4 synthesis was observed.

GTPases and Low Molecular Weight G Proteins during Cell-Cell Interaction between Neutrophils and Platelets

The involvement of low molecular weight G proteins (LMWG) was determined during cell-cell interaction between human neutrophils and platelets. α-[32P]-GTP binding revealed an enhanced expre

Signal Transduction and Priming of Human Neutrophils

Preincubation of human polymorphonuclear neutrophils (PMN) with diverse cytokines for 15 s enhanced the subsequent formyl-Met-Leu-Phe (FMLP)- induced leukotriene (LT) generation. These results correla

Basic Mechanisms of Cellular Priming and Release of Inflammatory Mediators

Simultaneous stimulation of human neutrophils (PMN) with the receptor-mediated activator formyl-met-leu-phe (FMLP) and the G protein activator sodium fluoride (NaF) resulted in the synergistic generation of leukotrienes. Activation of human platelets with thrombin and NaF showed an additive formation of 12-HETE. This enhancement in lipid mediator generation correlated with a time-dependent synergism in G protein activation after sequential stimulation of intact cells with FMLP and NaF or thrombin and NaF, respectively. In addition, polymerization of actin, an early event in cell activation, was enhanced after incubation with cytokines and FMLP.