A SELDI-TOF MS study of the genetic and post-translational molecular heterogeneity of eosinophil cationic protein.

Eosinophil cationic protein (ECP), a secretory protein of the eosinophil granulocyte, is a basic and highly heterogeneous protein. This heterogeneity is dependent on polymorphisms in the ECP gene and post-translational modifications, and it affects the functional properties of the protein in terms of cytotoxicity. The aim of this study was to further investigate the molecular heterogeneity, hence, an affinity capture assay based on an antigen-antibody interaction with the surface-enhanced laser desorption/ionization-time of flight mass spectrometry (SELDI-TOF MS) technique was developed. Of three monoclonal antibodies tested, that is, EG2, 614, and 652, the 614 mab was chosen for the experiments. ECP heterogeneity of single individuals was studied in extracts of purified blood eosinophils, and the presence of approximately 5 major molecular species was demonstrated in each subject. ECP from subjects with different ECP 434(G>C) genotypes (arg97thr) showed mass differences corresponding to the amino acid shift from arginine to threonine. ECP purified from pooled leukocytes of large numbers of healthy blood donors demonstrated an extensive mass heterogeneity with approximately 10 major molecular species. By the use of a variety of glucosidases it was shown that this heterogeneity was mainly due to N-linked oligosaccharides on which sialic acid, galactose, and acetylglucosamine was positioned. We conclude that the SELDI-TOF MS technique using specific monoclonal antibodies is a convenient and versatile tool; by means of this technique, we could detect both genetic and post-translational causes of the molecular heterogeneity of the eosinophil cationic protein.

Leukocyte CD11b and CD62l expression in response to interstitial inflammation in CAPD patients.

OBJECTIVE: Very little is known about the kinetics of leukocyte recruitment and the modulation of adhesion molecules on leukocytes in the interstitium at the site of inflammation outside the peritoneal cavity in patients on peritoneal dialysis. These issues were addressed in the present study. PATIENTS AND METHODS: Two skin blisters were raised in 10 patients on peritoneal dialysis and in 19 healthy subjects. After 12 hours, blister exudates were collected and the blisters were thereafter challenged with buffer or autologous serum in order to establish an intermediate and an intense cutaneous inflammation. Leukocyte count, leukocyte CD11b/CD62L expression, monocyte chemotactic protein-1 (MCP-1), and interleukin-8 (IL-8) were determined in blood and at the three sites of interstitial inflammation by immunostaining, flow cytometry, and ELISA. RESULTS: In monocytes and granulocytes, expression of CD11b increased and CD62L decreased significantly during the transmigration process from the peripheral blood into the three sites of interstitial inflammation. In the nonstimulated blister, expression of CD11b on both monocytes and granulocytes was similar in patients and healthy subjects. At the site of intermediate inflammation, expression of CD11b on both monocytes and granulocytes was significantly higher in healthy subjects compared to patients (p < 0.05 and p < 0.001 respectively). At the site of intense inflammation, expression of CD11b on granulocytes was significantly lower (p < 0.001), and CD62L significantly higher (p < 0.001) in patients. Interstitial concentrations of MCP-1 and IL-8 at the sites of intermediate inflammation were significantly higher in healthy subjects compared to patients (p < 0.05 and p < 0.05 respectively). However, at the site of intense inflammation, similar concentrations of MCP-1 and IL-8 were observed. Furthermore, there were no significant correlations between concentrations of MCP-1 and IL-8 in blister exudates and expression of CD11b on monocytes and granulocytes at the sites of interstitial inflammation. CONCLUSION: The ability of monocytes and granulocytes to modulate the expression of adhesion molecule in response to interstitial inflammation was significantly impaired in patients on peritoneal dialysis. Furthermore, these data suggest that the expression of CD11b on leukocytes is not dependent on concentrations of IL-8 and MCP-1 in interstitium in this patient group.

The autoantibody repertoire against copper- or macrophage-modified LDL differs in normolipidemics and hypercholesterolemic patients.

We have analyzed the antibody repertoire from normo- and hypercholesterolemic subjects to investigate how it can be related to macrophage-dependent modification of low-density lipoproteins, in comparison to the commonly used copper-oxidized LDL. Preexisting natural antibodies in plasma from normo- and hypercholesterolemic individuals were tested for their reactivity against copper ion oxidized LDL and LDL modified by macrophages. A crosswise comparison between these two antigen preparations demonstrated a different antibody repertoire in normo- and hypercholesterolemic patients. This study suggest that the search for antibodies that can influence the progression or regression of an atherosclerotic process has to take into account the process by which LDL is modified, and the repertoire of antibodies that is generated in the normal population, in comparison to that with, or at risk for, coronary artery diseases.

Unconventional strategies for the suppression of allergic asthma.

Allergic asthma results from an intrapulmonary allergen-driven Th2 response and is characterized by intermittent airway obstruction, airway hyperreactivity, and airway inflammation. An inverse association between allergic asthma and microbial infections has been observed. And this observation constitutes the base of the hygiene hypothesis. Here we discuss the hygiene hypothesis with emphasis on regulatory cells. We review the evidence for the emergence of regulatory cells, such as CD4(+)CD25(+) T cells during infection or during induction of tolerance by mucosal antigen administration. The review focuses also on the emergence of activated CD8(+) T cells and macrophages, induced by infections or microbial products, which also can result in the suppression of asthma. The underlying mechanisms by which regulatory immune cells suppress asthma may represent novel unconventional strategies controlling asthma.

Effects of fractions of traffic particulate matter on TH2-cytokines, IgE levels, and bronchial hyperresponsiveness in mice.

In recent decades an increased prevalence of allergic conditions has been observed in developed countries. Although lifestyles, exposure to infection, and diet are all likely important factors, many studies have also shown a strong link between industrialization and allergy. The aim of this study was to investigate which extract fractions from traffic particulate matter (TPM, collected in a tunnel in Prague) have the greatest impact on different inflammatory and immunological parameters, such as cytokine production, levels of immunoglobulin E (IgE) antibodies, and bronchial hyperresponsiveness (BHR) in mice, when the extracts are used together with birch pollen for immunization. BP2 mice were immunized with birch pollen and different fractions of TPM (fractions 1-8). They were provoked intranasally with a mixture of pollen and TPM or pollen alone before they were challenged with methacholine. The BHR was evaluated in a whole-body plethysmograph. Th2 cytokines and fibronectin concentrations were measured, and differential cell counts were performed in the bronchioalveolar lavage (BAL) fluid. Sera were collected for determination of antibody titers. The highest titers of IgE and the highest BHR were found in the positive control mice (immunized and provoked with a mixture of pollen and TPM), followed by mice immunized with pollen and fraction 2 (which contains organic acids). Fraction 2 also induced the highest number of eosinophils and increased levels of interleukin 5 (IL-5) in the BAL fluid. The highest levels of IL-5, in BAL fluid and sera, were obtained in mice immunized with fraction 6 (moderately polar compounds), a somewhat surprising result since those mice did not produce any IgE, did not have any eosinophils in their BAL, or showed almost no BHR. Our data demonstrate that fractions 2 (organic acids) and 7 (highly polar compounds) seem to contain potential adjuvants stimulating the IL-5 production, the IgE synthesis, the eosinophil recruitment, and the bronchial hyperreactivity. Further characterization at the molecular level is now necessary to be able to identify the exact nature of those potential adjuvants. This will be of help in the future to improve the quality of the urban air aerosol.

Hemodialysis-activated granulocytes at the site of interstitial inflammation.

It is not known to what extent intravascular phenotypic alterations in adhesion molecule expression induced by hemodialysis influence the recruitment of granulocytes and their ability to up-regulate CD11b at the local site of inflammation in the interstitium. We used a skin suction chamber technique to address this issue. Two skin blisters were raised on the forearm of eight hemodialysis patients and eight healthy subjects, and blister exudate was collected (time 0). The two blisters were stimulated with buffer (intermediate inflammation) or autologous serum (intense inflammation). Then the patients underwent cuprophane hemodialysis for 4 hours. Ten hours after start of dialysis, the exudate was aspirated from each chamber. Granulocyte count and surface expression of CD11b and CD62L were analyzed in samples from peripheral blood and blister exudate by flow cytometry. Granulocytes from healthy blood donors were incubated in blister fluid from patients and healthy subjects to determine the local chemotactic activity in terms of CD11b up-regulation. The expression of CD11b increased fourfold and CD62L decreased simultaneously in patients and healthy subjects when cells transmigrated to the unstimulated blister at time 0. At the site of intermediate inflammation, granulocytes from patients had a significantly lower capacity to mobilize CD11b compared with cells from healthy subjects (P < 0.001). At the site of intense interstitial inflammation, granulocytes from patients had the capacity to mobilize the receptor and reached values close to those obtained in healthy subjects (P = 0.079). The blister exudate from patients had a similar (at time 0 and intermediate inflammation) or higher (intense inflammation; P < 0.05) capacity to up-regulate CD11b on granulocytes in vitro compared with blister exudate from healthy subjects. Granulocytes from hemodialysis patients seem to require a more intense chemotactic stimulus to up-regulate CD11b at the local site of inflammation in the interstitium compared with corresponding cells from healthy subjects despite the fact that cells transmigrate in a milieu that contains chemotactic factors with an equal or higher capacity to up-regulate CD11b. Granulocytes in hemodialysis patients seem to be more refractory to inflammatory stimuli in the interstitium.