Chemiluminescence in inflammatory bowel disease patients: a parameter of inflammatory activity.

BACKGROUND: Reactive oxygen species (ROS) are produced in excess in the inflamed mucosa and peripheral blood of patients with inflammatory bowel disease. These species have emerged as a common pathway of tissue injury in a wide variety of inflammatory and other disease processes. The present study was conducted to assess ROS production and to correlate this with parameters of inflammatory activity. METHODS: In 25 patients with Crohn's disease (CD), 20 patients with ulcerative colitis (UC) and 65 age- and sex-matched healthy volunteers ROS production was measured using the whole blood luminol enhanced chemiluminescence assay (LECA). Disease activity was assessed using the Crohn's disease activity index and the Ulcerative Colitis Symptoms Score (UCSS) for CD and UC, respectively. Furthermore, the effect of various scavengers, enzymes and enzyme inhibitors on LECA was studied to assess the contribution of different ROS. RESULTS: LECA was significantly higher in CD and UC patients compared with healthy controls (7.1+/-4.7 and 9.8+/-6 vs. 5.2+/-2.8 x 10(3) counts per minute (cpm), p<0.05 and <0.001). In CD, relative LECA (patient/control) was correlated with the Crohn's disease activity index and C-reactive protein (CRP) (r=0.54, p=0.001 and r=0.51, p=0.01). In UC, CRP but not LECA was correlated with the Ulcerative Colitis Symptoms Score (C-reactive protein: r=0.42, p=0.01). Addition of azide, superoxide dismutase, deferoxamine and dimethylthiourea resulted in a decrease of LECA values. CONCLUSION: Whole blood LECA is increased in patients with CD and UC. This parameter is correlated with disease activity in CD. The observed chemiluminescence is probably due to generation of superoxide and the hydroxyl radical.

Granulocyte function in women with diabetes and asymptomatic bacteriuria.

OBJECTIVE: To study whether diabetic women with asymptomatic bacteriuria have impaired granulocyte function and compare them with nonbacteriuric diabetic women. RESEARCH DESIGN AND METHODS: A prevalence study with granulocyte function testing in a randomly selected number of patients was conducted; the setting was the university. The patients consisted of 63 women visiting the outpatient clinic for routine control of their diabetes. Measurements of routine blood controls and urine cultures were conducted in all patients. Granulocyte function testing (chemotaxis, opsonization, oxidative burst, phagocytosis, and killing) was performed in the first 20 patients (10 with and 10 without asymptomatic bacteriuria) and in 7 healthy control subjects. RESULTS: The prevalence of bacteriuria was 32%. Demographic characteristics were not significantly different between bacteriuric and nonbacteriuric women. Leukocytes were found more often in the urine of bacteriuric women (P < 0.05). No differences in any of the granulocyte function tests were documented among diabetic women with true asymptomatic bacteriuria, nonbacteriuric women, and healthy control subjects. CONCLUSIONS: The prevalence of asymptomatic bacteriuria is increased in women with diabetes. Granulocyte function impairment, however, cannot be the explanation for this finding.

Uncoupling of oxidative and non-oxidative mechanisms in human granulocyte-mediated cytotoxicity: use of cytoplasts and cells from chronic granulomatous disease patient.

Human polymorphonuclear leukocytes (PMN) and granule-free cytoplasts were compared for their cytotoxic capacities against red blood cells (RBC) and K562 tumor cells. Phorbol myristate acetate (PMA) stimulated PMN to efficient lysis of RBC targets, while cytotoxicity against the tumor cell line K562 was moderate. Activated cytoplasts also lysed RBC targets but were not able to kill K562 tumor cells, even at high cell numbers. Suppression of the glutathione redox cycle of the K562 tumor targets markedly increased their susceptibility to lysis by PMA-activated PMN. Despite the enhanced susceptibility of antioxidant-depleted K562 tumor cells to oxygen radical-induced damage, PMA-stimulated cytoplasts did not kill these targets. Addition of exogenous myeloperoxidase or lactoferrin to cytoplasts devoid of granule did not improve the lysis of RBC and K562 tumor cells. Coating K562 targets with specific antibodies induced efficient PMN-mediated killing in comparison to PMA-stimulated lysis of non-coated targets. Cytoplasts, however, did not kill antibody-coated K562 tumor cells; this was not improved by glutathione depletion but showed some lysis of antibody-coated RBC. PMN from a patient with chronic granulomatous disease (CGD) showed normal antibody-dependent cell-mediated cytotoxicity (ADCC) against K562 tumor cells but were not able to lyse these targets after PMA stimulation. The analysis of target cell killing by cytoplasts and PMN from a CGD patient indicated that granular constituents are important mediators in the killing of nucleated target cells and that PMN-mediated ADCC does not require the release of reactive oxygen species. Differences in the susceptibility of target cells to oxygen-mediated lysis indicates that target cell antioxidant mechanisms play an important role in the outcome of the cytotoxic response.

Quantitation of conjugate formation between human polymorphonuclear leukocytes and antibody-coated target cells by flow cytometry: the role of Fc receptor and LFA-1 antigen.

The specific binding of human polymorphonuclear leukocytes (PMN) to antibody-coated target cells was characterized by flow cytometry. PMN were labeled with phycoerythrin-E (PE) via a granulocyte-specific monoclonal antibody (leu-M1) and mixed with fluorescein isothiocyanate-labeled K562 tumor cells sensitized with rabbit antiserum. Specific conjugates were formed as analyzed by two-color fluorescence in a flow cytometer. The formation of stable conjugates was dependent on initiation of contact, temperature, time, and antiserum concentration. Studies with inhibitors implicate that microfilaments, but not microtubules, Ca2+, Mg2+, or energy-dependent processes were a prerequisite for binding of PMN to the antibody-coated target cells. No conjugates were formed when uncoated target cells were used or when the experiment was performed in the presence of protein A, indicating that binding was specifically mediated through Fc receptors (FcR). Monoclonal antibodies against the FcRII and FcRIII were used to address the role of these receptors in conjugation. One of the two anti-FcRIII antibodies and an anti-FcRII antibody effectively prevented conjugation. A monoclonal antibody directed against the common beta-chain of the adhesion molecule family and a combination of antibodies against the alpha-chain of LFA-1 and Mo-1 also blocked conjugation when target cells were sensitized under suboptimal conditions. The antibody against the beta-chain also diminished killing of antibody-coated K562, as measured by chromium release when included in the cytotoxicity assay. These results indicate that flow cytometry permits accurate quantitation and characterization of the binding between PMN and antibody-coated target cells, which in principle, can be prevented by monoclonal antibodies against surface receptors. Binding is primarily established by both the FcRII and FcRIII. Adhesion-associated molecules on the PMN surface contribute to optimal binding.

Antibody-coated target cell membrane-induced chemiluminescence by human polymorphonuclear leukocytes.

Activation of the oxidative metabolic burst of human polymorphonuclear leukocytes (PMN) by antibody-coated crude membrane fragments of K562 tumor cells was measured in a luminometer. Induction of the chemiluminescence (Cl) response was measured in the presence of luminol and lucigenin. The Cl was dependent on the concentration of PMN, the enhancer luminol or lucigenin, and the amount of tumor cell fragments and anti-K562 serum. PMN were not triggered to a Cl response in the absence of antibodies. The lucigenin-enhanced Cl involved superoxide anion detection while the luminol-enhanced Cl was dependent on the presence of myeloperoxidase and involved hydroxyl radicals. An intact cytoskeleton and energy were necessary for an optimal Cl response.

Further evidence against a role for toxic oxygen products as lytic agents in NK cell-mediated cytotoxicity.

Natural killer (NK) cells are implicated in host defense mechanisms against infectious diseases and malignancies, and exert a rapid spontaneous cytolysis of various tumour cells and virus-infected cells without prior sensitization or activation (Herberman & Ortaldo, 1981). Human NK cells are a subpopulation of non-adherent, non-phagocytic lymphocytes defined as large granular lymphocytes (Timonen, Ortaldo & Herberman, 1981). NK cells possess a receptor for the Fc region of IgG (Perussia et al., 1984) that enables them to attack antibody-loaded targets, a process called antibody-dependent cell-mediated cytotoxicity (ADCC). The cytotoxic reaction of NK cells can be described as a 'stimulus-secretion' model, divided into three definable steps: binding, triggering for lysis and a killer cell-independent lytic step (Hiserodt, Britvan & Targan, 1982). The killing reaction involves a Ca2+-dependent activation, cytoskeletal rearrangement, activation of the arachidonic acid cascade, release of lysosomal enzymes and a cytotoxic factor(s) (Henkart, 1985) and, possibly, production of reactive oxygen species (Helfand, Werkmeister & Roder, 1982; Roder et al., 1982). The role and involvement of reactive oxygen species is still controversial. To study a possible participation of toxic oxygen species in NK-cell mediated cytotoxicity, we altered target cell anti-oxidant defence mechanisms and measured spontaneous NK-cell mediated cytotoxicity and ADCC reactions against tumour cells. We showed that alteration of target cell anti-oxidant systems had no effect on target cell susceptibility to NK-cell mediated killing. In contrast, the susceptibility of the anti-oxidant-depleted targets to oxygen-dependent polymorphonuclear leucocyte (PMN)-mediated cytotoxicity was increased.

Measurement of antibody-mediated binding of human polymorphonuclear leukocytes to HSV-1 infected anchorage fibroblasts.

A method for the quantitation of effector cell binding to anchorage fibroblast monolayers infected with HSV-1 is described. Human peripheral blood polymorphonuclear leukocytes (PMN) as effector cells were labeled with chromium-51. Fetal human lung fibroblasts were grown to confluency in microtiter plates, infected with HSV-1 and loaded with anti-HSV antibody. The amount of radiolabeled PMN adhering to the monolayer was determined after appropriate incubation and washings. The effector binding assay was shown to be dependent on specific anti-HSV antibodies, antibody concentration, HSV viral expression, and inoculation time. This assay system is especially useful for the evaluation of effector to target cell conjugate formation when applied to anchorage target cells.