Leucine aminopeptidase of neutrophils and lymphocytes in patients with cancer

The leucine aminopeptidase activity has been determined by using the cytochemical method of Burston and Folk in peripheral blood neutrophils and lymphocytes of 45 patients with various malignancies. Lung cancer, carcinoma of the stomach and cancer of the colon was diagnosed in 24, 16, and 5 patients, respectively. Patients with metastases showed a significantly higher activity of the enzyme if compared with that in the control group of healthy subjects and patients without metastases. The percentage of enzyme-positive lymphocytes was elevated significantly in patients with metastases whereas a total percentage of lymphocytes with regard to differential leukocyte count was diminished both in patients with and without metastases. The absolute count of neutrophils was elevated both in patients with and without metastases. The authors discuss the significance of their observation with regard to the antitumor cytotoxic effect of neutrophils and lymphocytes.     

Polarized-light-stimulated enzymatic hydrolysis of xylan

The 1- to 2-h illumination of xylanase with visible polarized light (PL) prior to the action of that enzyme upon beechwood xylan significantly increased its activity. The activity only negligibly decreased on 3 months storage. The hydrolysis of xylan proceeded in three well-distinguished stages. In the first and fastest stage the effect of illumination was only slightly positive. The effect of the stimulation was noted in the second, slower stage. Enzyme stimulated with PL, preferably by means of the 2-h illumination, performed better than enzyme stimulated with nonpolarized light and non-stimulated enzyme. In the last, the slowest stage, the rates of the reaction were nearly the same using either stimulated or non-stimulated enzyme.     

Neutrophil function screening in patients with chronic granulomatous disease by a flow cytometric method.

Neutrophils from patients with chronic granulomatous disease (CGD) fail to produce a significant oxidative burst following stimulation. We have evaluated the use of flow cytometry and the dye 2',7'-dichlorofluorescein diacetate (DCF) for routine screening for deficiencies of neutrophil oxidative burst. A range for DCF fluorescence for phorbol myristate acetate stimulated and non-stimulated neutrophils was established based on data from 52 healthy adults. Samples from three patients with suspected neutrophil dysfunction, three patients with X-linked CGD, and one patient with autosomal recessive (AR) CGD were evaluated with both the DCF assay and the quantitative nitroblue tetrazolium dye reduction (NBT) test. For the DCF test, the ratio of mean fluorescence intensity of stimulated to non-stimulated neutrophils was less than 5 for CGD patients and from 16 to greater than 50 for healthy individuals. With the DCF test, two populations of neutrophils could be identified in samples from four carriers of X-linked CGD, although two carriers of AR CGD had NBT and DCF results in the normal range. Our data suggest the DCF test is a sensitive and convenient method for detecting CGD.     

NADPH:O2 oxidoreductase of human eosinophils in the cell-free system

The NADPH oxidase of human eosinophils, measured in the cell-free system, shows the same characteristics as the enzyme from human neutrophils. All proteins required for activity of the enzyme are expressed in eosinophils at a higher level than in neutrophils. Eosinophils isolated from patients with chronic granulomatous disease show the same molecular defects as the neutrophils from these patients.     

Deficiency of beta-glucuronidase in neutrophils from patients with precancerous states of the larynx.

Activity of beta-glucuronidase (GR) and acid phosphatase (AP) has been determined in peripheral blood neutrophils from 24 men with precancerous states of the larynx that is leukoplakia papillomas and pachydermia by means cytochemical methods described by Hayashi et al., and Barka and Anderson, respectively. The results obtained were expressed in terms of absolute counts of enzyme-positive and enzyme-negative cells with regard to enzyme activity variation within the enzyme-positive neutrophil population; the enzyme activity index score has been calculated. The control group consisted of 20 healthy subjects of the same sex. No significant alterations were found so far as AP activity is concerned between the group studied. In contrast, activity of GR in patients with precancerous states exhibited significant lowering. The most striking feature was in almost complete absence from the blood of GR-positive neutrophils with high activity of the enzyme. Majority of these cells showed only traces of the GR activity. According to authors opinion the deficiency of GR in neutrophils of patients with precancerous lesions pertains to problem of neutrophil-mediated cytotoxic effect against mammalian tumour cells.     

Localization of leukotriene D4-metabolizing metalloenzyme on the cell surface of human neutrophils

The localization of leukotriene D4-metabolizing enzyme on the cell surface was examined using human neutrophils. Intact neutrophils rapidly converted leukotriene D4 to leukotriene E4. However, when neutrophils were modified chemically by diazotized sulfanilic acid, a poorly permeant reagent which inactivates cell surface enzymes selectively, the leukotriene D4-metabolizing activity of neutrophils decreased significantly without any inhibition of the cell viability or marker enzymes of cytosol, granules, microsome and mitochondria. The leukotriene D4-metabolizing enzyme activity of the membrane fraction was inhibited by modification to the same extent as that of Mg2+-dependent ATPase, a cell-surface marker enzyme. Among various enzyme inhibitors examined, a metal chelator, o-phenanthroline, strongly suppressed the leukotriene D4-metabolizing activity of intact neutrophils and the o-phenanthroline-inactivated enzyme activity was fully reactivated by Co2+, Mn2+ and Zn2+. These results would suggest that some metalloenzyme located on the cell surface is involved in the conversion of leukotriene D4 to leukotriene E4 by neutrophils.     

Effects of mitogenic stimulation on lymphocyte alpha-D-mannosidases

Three types of alpha-D-mannosidase are present in human and murine lymphocytes. Their levels increased substantially when the cells were activated by T-cell mitogens, concanavalin A (Con A) and phytohaemagglutinin (PHA), and in the murine cells also by lipopolysaccharide (LPS), a B-cell mitogen. The intracellular localization of the alpha-D-mannosidases in the non-stimulated and activated murine cells was investigated by fractionation of lymphocyte lysates on colloidal silica (Percoll) and discontinuous sucrose gradients. In both types of cell, an enzyme having optimal activity at neutral pH was obtained in the cytosolic fraction and another alpha-D-mannosidase most active at an intermediate pH was obtained partly in membrane-bound form. In contrast, an acidic alpha-D-mannosidase, which was particularly elevated in the activated murine spleen cells, had a distribution in these lymphoblasts which was markedly different from that in non-stimulated lymphocytes. In the latter, the major proportion of the activity was obtained in a cytosolic fraction and the remainder in a particulate fraction of light density, whereas the enzyme in activated lymphocytes was distributed between vesicles of light and heavy density comparable with lysosomal organelles. Moreover, the acidic alpha-D-mannosidase still remained membrane bound even when cell lysates were prepared under hypotonic conditions which disrupt lysosome integrity. These results suggest that lymphocyte activation involves either stabilization of fragile lysosomes present in resting cells or de novo synthesis of lysosome-like structures. The acidic alpha-D-mannosidase present within isolated, intact lysosomes was found to be in a form, A, whereas a different form, B, was most prominent in whole-cell extracts of both types of lymphocyte.(ABSTRACT TRUNCATED AT 250 WORDS)     

Changes in the myeloperoxidase and acid phosphatase activities in human peripheral blood neutrophils with the cells stimulated in vitro]

In four series of experiments human peripheral blood neutrophils were found capable of synthetizing the active forms of such enzymes as myeloperoxidase and acid phosphatase after stimulation with opsonized zymosan, and the optimum conditions for testing the synthetizing activity of neutrophils were established. The examination of 39 practically healthy donors revealed an approximate equilibrium between the synthesis of the active forms of the enzyme and its excretion from the cell after this cell was activated. Considerable changes in the enzymatic activity of peripheral blood neutrophils were found in patients with yersiniosis, chronic herpes, chronic bronchitis and AIDS-associated complex. The possibility of a deficient enzymatic activity in some patients was suggested.     

Dependence of histochemical staining of leukocyte aminopeptidase upon its biochemical enzyme activity

The relationship between histochemical staining and biochemical activity of the enzyme was investigated using leukocytes with different aminopeptidase activities. In guinea-pig neutrophils and macrophages which have a relatively high enzyme activity, the histochemical staining correlated with the biochemical enzyme activity. On the other hand, guinea-pig lymphocytes and mouse neutrophils whose enzyme activities were 8.25 +/- 0.27 mU/10(7) cells and 6.18 +/- 0.87 mU/10(7) cells, respectively, were not stained by histochemical techniques. When guinea-pig neutrophils were modified chemically by diazotized sulfanilic acid at different concentrations, the histochemical staining of neutrophils decreased in proportion to the degree of inhibition of their biochemical enzyme activity and hardly became detectable below 10 mU/10(7) cells. However, guinea-pig neutrophils contained the soluble enzyme, corresponding to 5 mU/10(7) cells, which leaked out rapidly from cells during staining procedure, suggesting that the limit of visualization of the membrane-bound aminopeptidase activity by the histochemical techniques is about 5 mU/10(7) cells. The membrane-bound enzyme activities in guinea-pig lymphocytes and mouse neutrophils were 5 mU and 3 mU per 10(7) cells, respectively, and so it is possible that these leukocytes hardly stained histochemically.     

Beta-glucuronidase activity in neutrophils of patients with malignancies.

The human neutrophils induce cytotoxic effects on mammalian tumor cells. Hence it may be expected that intracellular enzymatic deficiencies of neutrophils may represent another cancer risk factor. In 205 patients with various malignancies the neutrophil beta-glucuronidase activity has been determined using a semiquantitative cytochemical method. A statistically significant deficiency of this enzyme in neutrophils has been observed in patients with precancerous states of the larynx, cancer of the larynx after radiotherapy and patients with cancer of large intestine. Patients with cancer of the lung and cancer of the stomach showed no changes with that regard whereas those with cancer of the breast demonstrated an increased enzyme activity.     

Differential synthesis of 5-lipoxygenase in peripheral blood and synovial fluid neutrophils in rheumatoid arthritis

In order to investigate 5-lipoxygenase enzyme regulation in neutrophils during an inflammatory reaction, we studied 5-lipoxygenase mRNA levels, as well as de novo enzyme synthesis, in resting and activated neutrophils isolated from normal individuals and patients with rheumatoid arthritis. The approach used was to analyze these activities in resting peripheral blood neutrophils of normal individuals on the one hand and in peripheral blood and matched synovial fluid neutrophils isolated from patients with rheumatoid arthritis on the other hand. Our first observation was that resting peripheral blood neutrophils of either normal individuals or patients show detectable levels of 5-lipoxygenase mRNA and are able to synthesize the enzyme de novo. Our second observation was that inflammatory activated neutrophils from synovial fluid reveal lower 5-lipoxygenase mRNA levels and enzyme synthesis than do the patient-matched peripheral blood cells. This is in spite of the fact that, for other proteins, synovial fluid neutrophils are equally or more active than their peripheral blood counterparts. We conclude that peripheral blood neutrophils are capable of synthesizing the enzyme, thus ensuring the turnover of the protein. Furthermore, complex regulatory mechanisms appear to take place in response to inflammation as it occurs in synovial fluids of patients with rheumatoid arthritis, leading to decreased mRNA levels and enzyme synthesis. Possible mechanisms of regulation are discussed and are presently under investigation.     

Changes in leukocyte enzyme activity during experimental amyloidogenesis and in patients with amyloidosis]

The activity of hydrolytic and redox myeloperoxidase enzymes was determined in the neutrophils and lymphocytes of the peripheral blood of patients with secondary amyloidosis and also of the animals with amyloidogenesis and amyloidosis (caseine model). During the amyloidogenesis the activity of hyocrolytic enzymes and of myeloperoxidase in the neutrophils was found to decrease; this was particularly marked at the stage of the initial amyloid deposition. Changes in the enzyme activity in the animals against the background of already developed amyloidosis coincided with such in the blood cells of patients with secondary amyloidosis. The results obtained are discussed from the aspects of resorption theory.     

Intracellular enzymatic response of lymphocytes and neutrophils in patients with cancer of the larynx.

In 20 men, aged 35 to 55 years, with untreated cancer of the larynx activity of lysosomal acid phosphatase (AP), beta-glucuronidase (GR) and N-acetyl-beta-glucosaminidase was determined cytochemically in peripheral blood lymphocytes and neutrophils by means of Barka and Anderson, Hayashi et al. and Hayashi's method, respectively; the results obtained were compared with those in 20 healthy men aged 20 to 30 years. Total count of GR-positive lymphocytes was higher in the patients than in normal persons. Total counts of AP-, GR-, and GS-positive lymphocytes with not disrupted enzyme-positive lysosomal granules within the cell cytoplasm were significantly lower and total counts of cells exhibiting the disruption of lysosomal granules and the diffuse type of cytochemical reaction were significantly higher in the patients when compared with the control group. The response of neutrophils consisted of a significant elevation in numbers of AP-, and GS-positive cells; overall score of enzyme activity studied in neutrophils was not altered in the patients. The authors disucss the significance of their observations in the light of data on participation of lymphocytic and neutrophilic lysosomal apparatus in the immunological response against tumour specific antigen in patients with cancer.     

Alcohol dehydrogenase activity in rat kidney cortex stimulated by oestradiol

Sex differences in alcohol dehydrogenase activity, determined by the influence of oestrogen hormones, were found to exist in the rat kidney. Oestradiol, but neither testosterone nor progesterone, was shown to be a powerful stimulator of kidney alcohol dehydrogenase activity in rats, maximally 6- to 8-times over control values. The Michaelis-Menten constant for acetaldehyde of both non-stimulated and oestradiol-stimulated kidney alcohol dehydrogenases was found to be similar, 6.7 · 10^?5 M and 7.8 · 10^?5 M, respectively. Actinomycin D was shown to have an additive effect (superinduction) on the oestradiol-induced increase in kidney enzyme activity. The findings suggest the possibility of the higher contribution of kidneys in ethanol metabolism in states with an elevated level of oestradiol, such as chronic ethanol intake and ethanol hepatic disease.     

Isolation and immunochemical localization of collagenase in mouse peritoneal macrophages

Collagenase was isolated from the culture medium of thioglycollate-stimulated mouse peritoneal exudate macrophages. The macrophage collagenase activity was inhibited by goat anti-mouse bone collagenase antibody, indicating that macrophage collagenase immunologically cross-reacts with mouse bone collagenase. The enzyme was localized in mouse peritoneal macrophages by indirect immunofluorescent antibody technique. Distinct granular fluorescence was observed intracellularly in most thioglycollate-stimulated macrophages, whereas slight or no fluorescence was observed in non-stimulated control macrophages.     

Comparative effects of two sulfhydryl reagents on the activities of a multifunctional red cell enzyme: bisphosphoglycerate mutase

The effects of two sulfhydryl reagents on the three activities of bisphosphoglycerate mutase have been compared. Under N-ethylmaleimide treatment all the activities were inhibited except for 60% of the non-stimulated phosphatase. With iodoacetamide the mutase and the stimulated-phosphatase activities were completely inhibited whereas the non-stimulated phosphatase and 60% of the synthase activities were unaffected. 2,3-bisphosphoglycerate protected all the activities of the enzyme against inactivation by the two sulfhydryl reagents whereas 3-phosphoglycerate protected them only against iodoacetamide. 2-phosphoglycolate had an identical effect to that of 3-phosphoglycerate except for its effect on the non-stimulated phosphatase activity, which was slightly enhanced under N-ethylmaleimide treatment.     

Oxidative Burst of Circulating Neutrophils Following Traumatic Brain Injury in Human

Besides secondary injury at the lesional site, Traumatic brain injury (TBI) can cause a systemic inflammatory response, which may cause damage to initially unaffected organs and potentially further exacerbate the original injury. Here we investigated plasma levels of important inflammatory mediators, oxidative activity of circulating leukocytes, particularly focusing on neutrophils, from TBI subjects and control subjects with general trauma from 6 hours to 2 weeks following injury, comparing with values from uninjured subjects. We observed increased plasma level of inflammatory cytokines/molecules TNF-α, IL-6 and CRP, dramatically increased circulating leukocyte counts and elevated expression of TNF-α and iNOS in circulating leukocytes from TBI patients, which suggests a systemic inflammatory response following TBI. Our data further showed increased free radical production in leukocyte homogenates and elevated expression of key oxidative enzymes iNOS, COX-2 and NADPH oxidase (gp91^phox) in circulating leukocytes, indicating an intense induction of oxidative burst following TBI, which is significantly greater than that in control subjects with general trauma. Furthermore, flow cytometry assay proved neutrophils as the largest population in circulation after TBI and showed significantly up-regulated oxidative activity and suppressed phagocytosis rate for circulating neutrophils following brain trauma. It suggests that the highly activated neutrophils might play an important role in the secondary damage, even outside the injured brain. Taken together, the potent systemic inflammatory response induced by TBI, especially the intensively increase oxidative activity of circulating leukocytes, mainly neutrophils, may lead to a systemic damage, dysfunction/damage of bystander tissues/organs and even further exacerbate secondary local damage. Controlling these pathophysiological processes may be a promising therapeutic strategy and will protect unaffected organs and the injured brain from the secondary damage.     

Characterization of hemoglobin-hydrolyzing acidic proteinases in human and rat neutrophils

The nature and levels of hemoglobin (Hb)-hydrolyzing acidic proteinases including cathepsin D and cathepsin E, which were most active at pH 3.5-4.0, were enzymatically and immunochemically compared between human and rat neutrophils. By subcellular fractionation and immunoprecipitation with discriminative antibodies specific for each enzyme, cathepsin D was shown to be present in the granular content fraction of both human and rat neutrophils and to account for about 35% of the total Hb-hydrolyzing activity. Cathepsin E was observed mainly in the cytoplasmic fraction of rat neutrophils from peripheral blood and peritoneal exudates and accounted for about 65% of the total activity, but it was not detected in human blood neutrophils. Immunoelectron microscopy on rat neutrophils revealed that cathepsin D was exclusively confined to lysosomes, whereas cathepsin E was localized mainly in the cytoplasmic matrix and often in the perinuclear spaces and the rough endoplasmic reticulum. The non-cathepsin D activity in human neutrophils, which represented about 65% of the total activity, appeared to be due to a serine proteinase, since it was inhibited by diisopropyl fluorophosphate and phenylmethylsulfonyl fluoride and was not inhibited by agents specific for aspartic-, cysteine-, or metallo proteinases. The enzyme(s) responsible for this activity was largely associated with the granular membranes, and a half of it could be described as an integral membrane protein on the basis of phase separation with Triton X-114 at 35 degrees C. The levels of these Hb-hydrolases in gingival crevicular fluid from human chronic inflammatory periodontitis patients were examined in order to clarify their participation in the periodontal tissue breakdown.(ABSTRACT TRUNCATED AT 250 WORDS)     

Relationship between oxidative burst activity and CD11b expression in neutrophils and monocytes from healthy individuals: effects of race and gender

Oxidative burst activity and the expression of adhesion molecules have been used as indicators of leukocyte activation status. The aim of the study was to delineate the relationship of oxidative burst activity and the expression of adhesion molecules in neutrophils and monocytes from a pool of healthy volunteers (n = 96). We also tested the potential role of gender and a racial background in the individual response differences. Basal and phorbol myristate acetate (PMA)-stimulated oxidative burst and CD11b expression were determined using dihydrorhodamine 123 and phycoerythrin (PE)-conjugated anti-CD11b monoclonal antibodies. PMA markedly increased CD11b expression and cellular oxidant content in neutrophils and monocytes in all samples. However, the responses showed considerable variability among individuals. A positive correlation was observed between the responsiveness of neutrophils and monocytes in their basal or PMA-stimulated CD11b expressions and PMA-stimulated oxidative burst activities. In contrast, no correlation was found between the level of adhesion molecule expression and cellular oxidant content in monocytes or neutrophils either under basal or under PMA-stimulated conditions. The reactivity of oxidative burst (i.e., PMA-stimulated over basal) was significantly lower in neutrophils from African American males compared with cells from African American females, white females, or white males. In contrast, reactivity of monocytes was significantly elevated in white males compared with all other groups. These findings indicate that leukocytes with a relatively high degree of adhesion molecule expression may display an average or decreased oxidative burst activity, and vice versa. Our findings also indicate that ethnic background may influence the oxidative burst activity in neutrophils and monocytes. This needs consideration in clinical studies utilizing healthy volunteers with mixed gender and ethnic backgrounds.     

Structure determination of a human lymphocyte derived neutrophil activating peptide (LYNAP)

Phytohemagglutinin or Concanavalin A-stimulated human T-lymphocytes produce a factor (LYNAP) with potent chemotactic and enzyme degranulating activity in peripheral human neutrophils. Sequence analysis of LYNAP established an apparently novel 72 residue polypeptide structure. Examination of protein data bases showed that LYNAP had about 30% sequence homology with recently characterised connective tissue activating proteins produced by platelets. Furthermore, it was subsequently found that the amino acid sequence is largely the same as that predicted from a cDNA clone derived from mRNA elevated in peripheral human leukocytes stimulated by mitogens.