Hemin,a heme oxygenase substrate analog,inhibits the cell surface expression of CD11b and CD66b on human neutrophils

BACKGROUND: Neutrophils are signaled to sites of infection and inflammation by different chemotactic stimuli. In order to reach the airways they have to adhere to, and then migrate through, the endothelium of pulmonary vessels. Carbon monoxide (CO) is a gaseous mediator, endogenously produced in the human airways. Increased CO production has been demonstrated during airway inflammation and CO as well as hemin, a substrate for CO producing enzymes, has been shown to affect neutrophil migration. Our objective was to investigate if the neutrophil cell surface expression of CD11b, CD66b and CD63 was changed during intermittent allergic rhinitis and to establish whether CO could affect the expression of these markers of cellular activation. METHODS: Blood from 10 healthy volunteers was drawn and incubated with different concentrations of hemin. Blood from 12 other healthy volunteers and from 12 patients with intermittent allergic rhinitis was also drawn during grass pollen season. Neutrophils were then isolated from all these three sets, and their expression of CD antigens measured using flow cytometry. RESULTS: Patients with symptomatic intermittent allergic rhinitis exhibited lower levels of CD11b and CD66b on the neutrophil cell surface. Incubation with hemin decreased the expression of CD11b and CD66b. CD63 was generally weakly expressed and not significantly affected by hemin incubation. CONCLUSION: Our results demonstrate that expressions of neutrophil cell surface glycoproteins are changed during the season in patents with intermittent allergic rhinitis and that hemin, a substrate for CO production, may act as an inhibitor of neutrophil activation. This indicates a possible role for CO in the immune defense system.     

Myeloperoxidase (MPO) as a marker of neutrophil influx into nasal mucosa after recombinant IL-8 challenge

Myeloperoxidase (MPO) has been proposed to mirror the degree of neutrophil activation, however its role as a marker of the participation of neutrophils in allergic inflammation is still unclear as the literature is controversial. The aim of this study was to evaluate the MPO levels and neutrophil influx in nasal lavage after recombinant Il-8 challenge. Eight patients suffering from seasonal allergic rhinitis, hypersensitive to grass pollens, with average age 30.1 +/- 2.67 were challenged both with Il-8 and diluent for Il-8 on a subsequent day, in two phases: before the pollen season (unprimed mucosa) and during the season (primed mucosa). Nasal lavage with saline were collected before, during Il-8 or placebo challenge and 30 minutes, 2 hours and 3 hours after the challenge. The number of neutrophils and MPO levels in the nasal fluid were determined. After the challenge with Il-8 of primed mucosa the number of neutrophils increased from 28250 cells/ml at the baseline to 28778, 251020 and 333660 at 0, 5, 2 and 3 hours respectively. There was a statistically significant difference between cells number after diluent and Il-8 challenge (p < 0.05, Wilcoxon rank-sum test). The number of neutrophils in the nasal lavage of primed mucosa after Il-8 challenge was significantly higher (p < 0.005) at all time points in comparison with the diluent and Il-8 challenges in the unprimed mucosa. There was no difference (p < 0.05) in MPO levels in the nasal lavage between Il-8 (mean 17.43 ng/ml +/- 10.98) and diluent challenge (20.98 ng/ml +/- 11.89) of unprimed mucosa. In the primed mucosa fluid we observed a peak of MPO level at 2 hours time point, however that was not significant as compared to diluent challenge (p = 0.465). We did not find the relationship between MPO levels and the neutrophils number in the lavage (rank Spearman factor, rs = 0.258, p = 0.42). Due to the lack of statistically confirmed relationship between MPO level and the number of neutrophils, MPO seems to be of little value as a marker of neutrophil influx into nasal mucosa.     

Recruitment of activated neutrophils correlates with disease severity in adult Crohn’s disease

Neutrophils are detected in inflamed colon in Crohn’s disease (CD). However, whether the frequency and/or activation of circulating or gut tissue neutrophils correlate with endoscopic severity remains to be investigated. A cohort of 73 CD patients was prospectively enrolled according to endoscopic severity and treatment history. Individuals with active disease were stratified using the Montreal classification. Harvey–Bradshaw Index (HBI) and Simple Endoscopic Score for Crohn’s Disease (SES-CD) were performed at the time of ileocolonoscopy. Frequency of neutrophils and their expression of CD66b and CD64 were assessed in paired blood and colonic biopsies using flow cytometry. The percentage of neutrophils increased in inflamed colon and correlated with SES-CD in the entire cohort of patients examined, as well as in the subgroup with inflammatory (B1) active disease. SES-CD further correlated with neutrophil CD66b expression in mucosa but not blood and, conversely, with neutrophil CD64 expression in blood but not mucosa. However, the evaluation of neutrophil activation in mucosa when compared to blood reflected disease activity more clearly. Finally, a neutrophil activation power index (CD66b in mucosa X CD64 in blood) that correlated with SES-CD discriminated between patients with mild and severe disease. In conclusion, the frequency and activation of colonic neutrophils correlated with SES-CD, highlighting that mucosal neutrophils are associated with disease severity in CD.     

The effect of recombinant interleukin-8 on eosinophils' and neutrophils' migration in vivo and in vitro

BACKGROUND: Interleukin-8 (IL-8) is a chemokine that causes chemotaxis of neutrophils, eosinophils and lymphocytes in vitro; however, its role as a chemoattractant in allergic inflammation is unclear. The objective of this study was to investigate the effect of nasal instillation of IL-8 on the influx of inflammatory cells. METHODS: Twelve patients suffering from seasonal allergic rhinitis hypersensitive to grass pollens, with average age 30.1 +/- 2.67 years were challenged both with diluent for IL-8 and IL-8 on a subsequent day, in two phases: before the pollen season (unprimed mucosa) and during the season (primed mucosa). The number of neutrophils, eosinophils and myeloperoxydase (MPO) levels in the nasal fluid collected after IL-8 or placebo challenge were determined. RESULTS: Challenge with IL-8 of primed nasal mucosa induced a significant influx of neutrophils (29 x 10(4) cells/ml at 0.5 h, 251 x 10(4) at 2 h and 334 x 10(4) at 3 h). Number of eosinophils in comparison with diluent challenge was not significant. There was no difference in MPO levels in the nasal lavage between IL-8 and diluent challenge of unprimed and primed mucosa. We did not find the relationship between MPO levels and the neutrophils number in the lavage (rank Spearman factor, RS = 0.258, P = 0.42). CONCLUSION: We have demonstrated that IL-8 causes influx of neutrophils but not eosinophils into nasal mucosa in vivo. MPO level seems to be of little value as a marker of neutrophil influx into nasal mucosa.     

Mechanism of topical glucocorticoid treatment of hay fever: IL-5 and eosinophil activation during natural allergen exposure are suppressed, but IL-4, IL-6, and IgE antibody production are unaffected

BACKGROUND: Allergic rhinitis is traditionally defined as an IgE- and mast cell-mediated hypersensitivity reaction. Allergen challenge models suggest that cytokines and eosinophil mediators may also play roles. However, the causal relationship among inflammatory cells, their products, and patients' symptoms during natural allergen exposure has not been established. OBJECTIVE: We sought to elucidate the mechanisms of seasonal allergic rhinitis and the beneficial effects of topical glucocorticoids. METHODS: Thirty patients with ragweed-induced hay fever and a strongly positive serologic test response for ragweed IgE antibody received budesonide nasal spray or placebo in a randomized, parallel, double-blind study. Nasal wash fluids and sera were collected before and during the hay fever season. The levels of inflammatory mediators and allergen-specific immunoglobulins were measured by immunoassay. The activation markers on blood eosinophils were quantitated by flow cytometry. RESULTS: Compared with placebo-treated patients, budesonide-treated patients had strikingly reduced symptoms. In the placebo group, nasal symptoms correlated with nasal lavage fluid eosinophil-derived neurotoxin and IL-5 levels. At the season peak, the budesonide-treated group had significantly lower nasal fluid eosinophil-derived neurotoxin, IL-5, and soluble intracellular adhesion molecule-1 levels. In the treated group eosinophil expression of CD11b was suppressed at the season peak. In contrast, levels of IL-4 and IL-6 in nasal fluid and the seasonal increases in serum ragweed-specific IgE and nasal fluid IgA antibodies did not differ between groups. CONCLUSION: Eosinophilic inflammation plays a critical role in seasonal allergic rhinitis symptoms. One of the therapeutic effects of glucocorticoids is to suppress this inflammation.     

Elevated secretion of myeloperoxidase by neutrophils from asthmatic patients: the effect of immunotherapy

BACKGROUND: There is increasing evidence of neutrophil participation in asthma and the allergic process. After activation, neutrophils release myeloperoxidase (MPO) together with other granule enzymes. OBJECTIVES: In this study we attempted to evaluate the release of MPO in vitro by neutrophils from asthmatic patients and the relationship between neutrophil degranulation and lung function, measured as FEV(1), of the patients. We also investigated the possible role of immunotherapy in the release of MPO by neutrophils. METHODS: Neutrophils were stimulated with formyl-methionyl-leucyl-phenylalanine for 45 minutes at 37 degrees C. MPO released from neutrophils was assayed by using an MPO enzyme immunoassay. RESULTS: Neutrophils released statistically significantly higher MPO levels in the asthmatic patients not receiving immunotherapy than in the healthy group. A significant inverse correlation was observed in the asthmatic group not receiving immunotherapy between MPO secretion and lung function, measured as FEV(1), of the patients. Neutrophils of the asthmatic group receiving immunotherapy released significantly less MPO than did those of the asthmatic group not receiving immunotherapy, with MPO levels equal to those from nonallergic subjects. CONCLUSIONS: We conclude that neutrophils obtained from allergic asthmatic patients have an increased propensity to release MPO. The experiments described here provide evidence that there is a significant inverse relationship between levels of MPO released by neutrophils from allergic patients and lung function, as assessed by FEV(1). Our study suggests that immunotherapy actively modifies the release of MPO in vitro by neutrophils from allergic asthmatic patients.     

l-Selectin expression on neutrophils from allergic patients

BACKGROUND: L-selectin (CD62L) is an adhesion molecule involved in leucocyte attachment to endothelium at sites of inflammation, and it has been demonstrated that L-selectin is rapidly shed after neutrophil activation. Recently, it has been reported that there is increasing evidence of neutrophil participation in asthma and the allergic process. OBJECTIVE: The present study was designed to determine whether an IgE-dependent mechanism can modulate L-selectin expression on the surface of neutrophils. Moreover, we analyse the potential implication of intracellular signal-transduction pathways and whether specific immunotherapy (IT), glucocorticoids and antihistamines might regulate this process. METHODS: Peripheral blood neutrophils from three groups of donors (asthmatic group without IT treatment, IT-treated asthmatic group and healthy group) were used. Cells were challenged in vitro with the specific allergen that produced clinical symptoms in asthmatic patients and also with the allergen to which the patients were not sensitive. Neutrophils from healthy donors were also challenged with allergens. Expression of CD62L on the neutrophil surface was analysed by flow cytometry, and soluble CD62L (sCD62L) in culture supernatant by ELISA. In an attempt to discover which IgE receptor is involved, we also challenged the neutrophils with monoclonal antibody to FcepsilonRI, FcepsilonRII (CD23) and galectin-3 receptors. RESULTS: When neutrophils from allergic patients were challenged with specific allergens that produce clinical allergy symptoms, L-selectin was down-regulated from the surface of those cells, accompanied by a concomitant up-regulation of soluble L-selectin in the supernatant. The challenge with antibodies against FCepsilonRI, FCepsilonRII (CD23) and galectin-3, induces down-modulation of L-selectin on the surface of the neutrophils in all three cases. Calphostin C, wortmannin and manoalide attenuated CD62L down-regulation, suggesting the potential implication of protein kinase C, phosphatidylinositol 3-kinase and phospholipase A(2) in the process. IT and glucocorticoids modulated allergen-dependent CD62L down-regulation, whereas antihistamines (terfenadine, loratadine and cetirizine) or nedocromil sodium did not affect the shedding of L-selectin. CONCLUSIONS: We present evidence that the neutrophil surface expression of CD62L can be modulated by an allergen-dependent mechanism. The modulation of CD62L expression can be induced through the three receptors of IgE. This process can be affected by IT.     

Systemic Up-Regulation of TLR4 Causes Lipopolysaccharide-Induced Augmentation of Nasal Cytokine Release in Allergic Rhinitis

Background: Allergic rhinitis is a systemic disorder, and it is clinically well recognized that it can be aggravated by infection. Activation of the innate immune system constitutes a critical element in the process. Toll-like receptors (TLRs) comprise a part of the innate immune system, and lipopolysaccharide (LPS)-induced activation of TLR4 represents bacterial-induced interactions in various model systems. The present study examines how TLR2 and TLR4 expression is affected by symptomatic allergic rhinitis, and if LPS added upon allergen affects nasal cytokine release. Methods: In patients with pollen-induced allergic rhinitis and healthy non-allergic volunteers, nasal lavage (NAL), peripheral blood and bone marrow were sampled before and during the pollen season. TLR2 and TLR4 expression was determined flow cytometrically. Changes in the TLR receptor expression pattern were evaluated by a nasal challenge with allergen followed by LPS, or vice versa. Symptoms along with cells and cytokines in NAL were analyzed. Results: TLR4 expression increased in leukocytes in NAL, peripheral blood and bone marrow during symptomatic allergic rhinitis. A similar increase was seen for TLR2 in neutrophils in blood. Nasal challenge with allergen followed by LPS augmented the release of IL-4, IL-5, IL-10, IL-13, IFN-γ and TNF-α. Conclusion: A systemic up-regulation of TLR4 in symptomatic allergic rhinitis may explain why LPS preceded by allergen increases nasal cytokine release.     

Effect of seasonal allergen exposure on mucosal IL-16 and CD4+ cells in patients with allergic rhinitis

BACKGROUND: CD4+ T cells constitute a major source of cytokines in allergic diseases such as allergic rhinitis. Interleukin (IL)-16 selectively recruits CD4+ cells. METHODS: We evaluated the effect of natural allergen exposure during a grass-pollen season on IL-16 expression and number of CD4+ cells in nasal mucosa. Patients with allergic rhinitis (n=16) were treated with either a nasal glucocorticoid beclomethasone (BDP; 400 microg/day) or placebo, and gave nasal biopsies prior to and during the grass-pollen season. The evaluated markers in allergic rhinitis patients were also compared to those in healthy control subjects (n=5). RESULTS: Prior to the pollen season, the expression of IL-16, but not the number of CD4+ cells, was significantly higher in patients with allergic rhinitis than in healthy control subjects. The grass-pollen season further increased IL-16 expression and also increased the number of CD4+ cells in placebo-treated, but not in BDP-treated, allergic rhinitis patients. The pollen-season-induced change in IL-16 expression and in CD4+ cells was significantly more pronounced in placebo- than in BDP-treated patients. There was a significant correlation between the change in IL-16 expression and the number of CD4+ cells. CONCLUSIONS: These data suggest that local upregulation of IL-16 expression contributes to the inflammation observed in seasonal allergic rhinitis. Hypothetically, inhibition of IL-16 expression can be one of several mechanisms by which nasal glucocorticoids achieve their anti-inflammatory effect in allergic rhinitis.     

Increased adhesion to vascular cell adhesion molecule-1 and intercellular adhesion molecule-1 of eosinophils from patients with asthma

Adhesion of peripheral blood eosinophil and neutrophil granulocytes to the endothelial cell adherence receptors E-selectin, vascular cell adhesion molecule-1, and intercellular adhesion molecule-1 has been measured. The study included patients with allergic rhinitis, patients with mild allergic and nonallergic asthma, and healthy individuals; 10 persons were in each group. In addition, assay of eosinophil and neutrophil cell surface expression of the receptor complex CD11b/CD18 was performed. Increased eosinophil adhesion to vascular cell adhesion molecule-1 (p < 0.05) and intercellular adhesion molecule-1 (p < 0.05) was demonstrated in the patients with a more labile asthma, that is, a peak expiratory flow rate variability of more than 10%, suggesting a relationship to the degree of ongoing inflammation in the airways of the patients. The increased eosinophil adhesion was most probably due to a functional upregulation of the CD11b/CD18 and very late activation antigen-4 receptors, because the number of receptors measured as cell surface expression was unaltered. The increased eosinophil adhesion in the patients with high peak expiratory flow rate variability appeared independent of atopy. The increased adhesion was not entirely specific to the eosinophils, because neutrophils from patients with a peak expiratory flow rate variability of more than 10% also demonstrated increased adhesion to intercellular adhesion molecule-1 (p < 0.05) when compared with neutrophils from the patients with low peak expiratory flow rate variability. In conclusion, the demonstrated priming of eosinophil adhesion to vascular cell adhesion molecule-1 and intercellular adhesion molecule-1 might be one contributing mechanism behind the selective accumulation of eosinophils in the lung tissue of patients with asthma. (J ALLERGY CLIN IMMUNOL 1995;96:941-50.)     

Cellular kinetics of an allergic‐type response in a sheep mammary gland model of inflammation

BACKGROUND: Tissue recruitment of eosinophils and activated lymphocytes is a characteristic feature of allergic reactions. However, little is known about the involvement of specific adhesion molecules in the traffic of leucocytes during the allergic response. OBJECTIVE: To use a sheep mammary infusion model to characterize the kinetics of cell recruitment and expression of cellular adhesion molecules and activation markers on eosinophils and lymphocytes involved in an allergic-type response. METHODS: Mature non-lactating ewes were primed and challenged by direct infusion of the mammary glands with nematode larvae. Using a non-invasive method of saline infusion and 'milking' of the glands, large numbers of inflammatory cells were repeatedly sampled over 10 to 96 h following their migration into the mammary gland lumen, and analyzed by 2-colour flow cytometry. RESULTS: Leucocyte recruitment into the mammary lumen was characterized by two separate phases involving an acute neutrophilic response at 10 h post-challenge, followed by a dramatic reduction in neutrophils and appearance of eosinophils and activated lymphocytes. From 48 h post-challenge, eosinophils were predominant and represented 40 to 65% of leucocytes in the mammary lavage (MAL). Increases in activated CD4+ T cells and gammadelta+ T cells were also observed at this time-point. The kinetics of expression of cell surface molecules on eosinophils and lymphocytes in blood and MAL were compared during the course of the allergic-type reaction. Adhesion molecule expression on lymphocytes was modulated following allergen challenge and an activation of MAL vs. blood lymphocytes was seen during the later stages of the allergic response. Eosinophil expression of VLA-4 and l-selectin was down-regulated compared with blood at all time-points examined. There were high levels of expression of CD11b and CD44 on eosinophils during the early compared to the late-phase of the allergic reaction. CONCLUSION: These results indicate the existence of two separate mechanisms of eosinophil recruitment during the allergic inflammatory response.     

Characterization of eosinophils and detection of eotaxin in skin chamber fluid after challenge with relevant allergen in patients with mild asthma

BACKGROUND: A selective recruitment of eosinophils to sites of allergic inflammation is suggested to be controlled by regulation of cytokines, chemokines and adhesion molecules. OBJECTIVE: The aim of this study was to examine whether allergen challenge in skin chambers, applied on patients with allergic rhinitis and mild asthma, results in a selective influx of activated eosinophils and detectable levels of cytokines/chemokines related to eosinophil recruitment, such as interleukin (IL)-5 and eotaxin. METHODS: A skin blister was induced on the volar aspect of each forearm; one contained PBS-heparin buffer (control) and the other was challenged with relevant allergen. Peripheral blood was drawn before the allergen was applied to the skin chamber, and the expression of CD9, CD11b and EG2-epitope on intracellular eosinophil cationic protein (ECP) was analysed in eosinophils. Chamber fluid was collected 8 h after allergen application and analysed for differential cell counts, expression of eosinophil activity markers, the presence of ECP, eotaxin, and IL-5. RESULTS: The number of recruited leucocytes was equal in the allergen-challenged chambers and in controls. However, the number of eosinophils was significantly increased in the allergen-challenged chambers, and elevated levels of released ECP were measured. Moreover, the eosinophils recruited were activated, as shown by increased expression of EG2 and CD11b, and decreased expression of CD9, in comparison with blood eosinophils. In the skin chamber fluids, higher levels of eotaxin were detected in the allergen-challenged chambers than in controls, but there were no detectable levels of IL-5. CONCLUSION: We have demonstrated a selective recruitment of eosinophils, and higher levels of released ECP and eotaxin, in skin chambers stimulated with allergen, as compared with control chambers. Allergen challenge in skin chambers is a useful tool for studies of eosinophil recruitment, their state of activation, and their involvement in the allergic inflammatory response.     

Neutrophils develop rapid proinflammatory response after engulfing Hb-activated platelets under intravascular hemolysis

Neutrophils maintain immune homeostasis by engulfing apoptotic cells and debris. We describe the rapid activation of neutrophils after engulfing hemoglobin (Hb)-activated platelets, which are abundant in the circulation of hemolytic patients. Neutrophils from healthy individuals after engulfing Hb-activated platelets express elevated CD11b and secrete significant amounts of tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6, myeloperoxidase (MPO) and elastase within 4-h platelets, but not with free-Hb only in vitro. These neutrophils exhibit early onset of apoptosis and cell death after engulfing Hb-activated platelets, but not with free-Hb only. Further, our data from mice with phenylhydrazine-induced intravascular hemolysis display a gradual decrease in total neutrophil count, but the number of activated neutrophils and neutrophil–platelet aggregates increases, along with the rise of TNF-α, IL-1β, IL-6 and MPO in circulation. Our data from paroxysmal nocturnal hemoglobinuria (PNH) patients confirmed the observation of decreased total neutrophil counts, but elevated numbers of activated neutrophils, including neutrophil–platelet aggregates, in parallel with elevated expression of TNFA, IL1B and IL6 genes in neutrophils, also increased levels of these cytokines along with MPO in circulation, and this correlated directly with elevated intravascular hemolysis (high free-Hb in plasma). The patients’ neutrophils displayed significant localization of intracellular Hb and platelets, unlike the counterparts from healthy individuals. Together, therefore, our observations suggest that Hb-activated platelets, which are abundant in the circulation of patients with hemolytic disorders, including PNH, promotes early onset of neutrophil activation and increases their proinflammatory response and leads to early apoptosis and cell death.     

Conditions in blood sampling procedures that extend the ex vivo stability of eosinophil activity markers in peripheral blood from allergic patients and healthy controls.

BACKGROUND: Serum-ECP, EG2-epitope on intracellular ECP and surface expression of CD9 and CD11b in peripheral blood eosinophils (PBE) are considered to be markers that mirror clinical parameters in allergic inflammation. OBJECTIVE: The aim was to investigate the impact of the blood sampling procedure on PBE markers and to identify optimal conditions for extended pre-analysis storage. METHODS: Blood, from healthy individuals and patients with allergic rhinitis/asthma, was collected in tubes with EDTA, citrate, or without anti-coagulant. The expression of EG2-epitope, CD9, and CD11b were analyzed in eosinophils and neutrophils after 1, 5, and 24 hours of storage at +4 degrees C, according to the FOG-method and flow cytometry. In vitro stimulation with fMLP/PMA was used for metabolic activity analysis and CD11b mobilization. Following a 1-hour clotting period at +20 to 22 degrees C, samples were stored at +4 degrees C and serum-ECP levels were measured. RESULTS: The EG2-epitope, serum-ECP, and CD9 were stable in samples from both healthy controls and allergic patients at all storage conditions. The EG2-epitope, serum-ECP and PBE count were significantly increased in the patient group, whereas no differences were observed in the expression of CD9 or CD11b. Both granulocytes and monocytes retained their metabolic activity for 24 hours. Neutrophils in citrate-blood increased their ability to respond to fMLP, as compared with EDTA-blood. CONCLUSION: In vitro analysis of selected activity markers and functional tests could be performed on granulocytes from both healthy individuals and allergic patients after 24 hours storage at +4 degrees C. The anticoagulant citrate seems to be preferable to EDTA when monocytes or CD11b expression are analyzed.     

The degree of natural allergen exposure modifies eosinophil activity markers in the circulation of patients with mild asthma

We have previously found that CD9, CD11b, and intracellular ECP (EG2) may be used as activity markers for eosinophils in vitro . The main object of the present study was to determine whether these markers can reflect eosinophil activation in vivo in relation to allergen exposure. For this purpose, six patients with a history of allergic rhinitis and occasional asthma symptoms during the pollen season participated. Blood donors served as controls. Peripheral blood eosinophils were analyzed according to the FOG method and flow cytometry, before and during one birch pollen season with high pollen load (HPL) and one with low pollen load (LPL). The CD9 expression on peripheral eosinophils from the patients was significantly increased both before (P<0.05) and during (P<0.01) HPL season, and CD11b expression solely during HPL season (P = 0.01) as compared to controls. The intracellular expression of the EG2 epitope was increased before (P<0.01) and during (P<0.05) HPL season, and increased significantly (P<0.05) during season as compared to before. No changes were observed before and during LPL season. The proportion of eosinophils was increased both before (P<0.05) and during (P<0.001) the HPL season as compared to controls. The markers CD9, EG2, and, to a lesser extent, CD11b seem to detect activated eosinophils in the circulation, whereas EG2 may also reflect increased antigen exposure during season.     

Neutrophil expression of tumour necrosis factor receptors (TNF-R) and of activation markers (CD11b, CD43, CD63) in rheumatoid arthritis.

In vitro analysis of polymorphonuclear neutrophils (PMN) has allowed various stages of cell activation to be distinguished, characterized by the expression level of specific membrane markers and of functional receptors. Among those, TNF-alpha receptors (TNF-R) are modulated by various PMN activators, a mechanism which may be important to control cell responses to TNF in inflammatory reactions such as rheumatoid arthritis (RA). PMN, isolated from the blood of 36 RA patients and from the synovial fluid of 23 of them, were analysed for membrane expression of the two TNF-R (p55 and p75). Soluble p55 and p75 (sTNF-R) and TNF concentrations were measured in the plasma and synovial fluid by specific ELISA assays. Our results show that PMN from the blood of RA patients bear a normal number of TNF-R, with a normal p55/p75 ratio, compared with PMN from normal controls. Soluble TNF-R levels were similar in patients and normal plasma. In spite of high endogenous TNF concentration, patients' circulating PMN were not activated, as shown by a CD11b/CD18 expression similar to that of control resting cells. In contrast with blood neutrophils, PMN from RA patients' synovial fluids had an activated phenotype, characterized by increased expression of CD11b, decreased expression of leukosialin, CD43, and the appearance on the plasma membrane of an azurophil granule protein, CD63. High levels of soluble TNF-R were measured in RA synovial fluids. Nevertheless, membrane TNF-R levels and p55 and p75 proportions were similar to those of PMN from normal blood. These results suggest the existence of regulatory mechanisms which maintain a stable neutrophil expression of TNF-R as well as a balance between both types of receptors in inflammatory situations where neutrophils are strongly activated.     

Interleukin-8 secretion in patients with allergic rhinitis after an allergen challenge: interleukin-8 is not the main chemotactic factor present in nasal lavages

Background Interleukin-8 (IL-8) is a chemotactic cytokine for neutrophils and primed eosinophils. In allergic rhinitis, allergen exposure triggers leucocyte recruitment. Objective We evaluated in this study IL-8 secretion and the neutrophil chemotactic activity in nasal lavages collected after a nasal allergen challenge. Moreover, the participation of IL-8 in the neutrophil chemotactic activity was quantified. Methods Four healthy subjects and 19 patients with allergic rhinitis were exposed to a nasal allergen challenge. As a control, saline challenge was performed in four patients with allergic rhinitis. Concentration of IL-8 was measured hy ELISA in nasal lavages collected before and after challenge. Neutrophil chemotactic assay was developed using a 48-well chemotaxis microassembly. Results After allergen challenge, the healthy subjects, the four patients receiving saline and one patient exposed to allergen did not respond; seven patients presented a single early reaction and 11 patients a dual response. For healthy subjects and the four patients exposed to saline, the level of IL-8 did not increase after challenge in comparison with that at baseline. After allergen challenge, two peaks of IL-8 release were observed for patients with allergic rhinitis during the early (30 min to 1 h 30 min) and the late periods (3 h 30 min to 9 h 30 min), however the difference was not significant for the early period. During the late period, a significant increase in IL-8 concentrations was detected for the patients developing a dual response, whereas the difference was not significant for those presenting only an early reaction. The neutrophil chemotactic activity of nasal lavages from patients with allergic rhinitis collected during the early and the late reactions (17 ± 2.1 and 23.3 ± 2.8 neutrophils per high power field (hpf), respectively) was significantly higher than the activity of lavage fluid collected at haseline (9.2 ± 1.8 neutrophils per hpf). Nevertheless, the addition of a neutralizing anti-IL-8 antibody inhibited weakly the chemotactic activity of lavage fluid from rhinitic patients collected during the early or the late periods (18 and 11% of inhibition) (P= NS). Conclusion These data show that allergen challenge increased significantly the secretion of IL-8 for the patients with allergic rhinitis. However, neutralization of IL-8 in nasal lavages by a specific antibody revealed that the role of this chemokine in granulocyte infiltrate was limited, suggesting that IL-8 acts in connection with other chemotactic factors in this recruitment.     

IgE-dependent release of myeloperoxidase by neutrophils from allergic patients

BACKGROUND: The three forms of IgE receptor: the heterotrimeric high-affinity receptor for IgE (FcepsilonRI), the low-affinity receptor for IgE (FcepsilonRII/CD23) and the Mac-2/IgE-binding protein (epsilonBP), have previously been found on human neutrophils. We have previously shown that specific allergens are able to activate functional responses by neutrophils from allergic patients sensitized to those allergens. Neutrophils are present in the sites of allergic inflammation. The primary (azurophilic) granules of neutrophils contain a variety of enzymes that might potentiate inflammation, such as myeloperoxidase (MPO). It is not known whether specific allergens are able to elicit MPO release by neutrophils from allergic patients. METHODS: Neutrophils were challenged in vitro with the specific allergen that produced clinical symptoms in asthmatic patients. Also, the cells were challenged with allergens that the patients were not sensitive to. Neutrophils from normal subjects were also challenged with allergens. RESULTS: The in vitro challenge of neutrophils with allergens that the patients were sensitive to elicited a release of MPO by these cells. The in vitro activation of neutrophils was highly allergen-specific, in such a way that allergens other than those accounting for clinical symptoms did not evoke MPO release, and allergens were ineffective on neutrophils from healthy donors. CONCLUSION: An IgE-dependent mechanism might promote MPO release by neutrophils at allergic sites.     

Immunocytologic analysis of nasal cells obtained by nasal lavage: a comparative study with a standard method of cell identification

For evaluation of two methods of nasal cell identification, cell morphology and immunocytologic analysis, nasal lavage was performed in 16 healthy subjects and 29 patients suffering from rhinitis. Nasal lavage smears were stained with May-Grünwald-Giemsa (MGG), and cells were identified according to their structure as epithelial cells, neutrophils, lymphocytes, eosinophils, and metachromatic cells (basophils and mast cells). Immunocytologic analysis was performed with monoclonal antibodies by the immunoalkaline phosphatase method. The following monoclonal antibodies were used: CK1, EG2, and CD3, which identify epithelial cells, activated eosinophils, and T lymphocytes, respectively; CD 15, which recognizes mature granulocytic cells; and CD 14, which reacts with monocytes and macrophages. A significant difference was observed between the two methods in the number of identified epithelial cells, in both controls (64.6 ± 7.8% with MGG, 14.2 ± 3.5% with CK1 analysis) and patients with rhinitis (56.9 ± 7.6% with MGG, 18.2 ± 3.7% with CK1 analysis). In contrast, no significant differences were found in eosinophil and neutrophil counts when the two methods were compared. After nasal allergic provocation, a significant increase in the number of eosinophils was observed with both methods in seven patients with rhinitis. The results of this study indicate that: 1) MGG staining is a useful method to identify the cells obtained by nasal lavage, and 2) immunocytologic analysis with monoclonal antibodies accurately identifies granulocytic cells, while only a low proportion of epithelial cells are detected, probably because anticytokeratin monoclonal antibody reacts only with viable cells.     

Inefficiency of C3H/HeN Mice to Control Chlamydial Lung Infection Correlates with Downregulation of Neutrophil Activation during the Late Stage of Infection

We previously reported that massive infiltration of neutrophils in C3H/HeN （C3H） mice could not efficiently control Chlamydia muridarum （Cm） infection and might contribute to the high susceptibility of these mice to lung infection. To further define the nature of neutrophil responses in C3H mice during chlamydial infection, we examine the expression of adhesion molecules and CDllb related to neutrophils infiltration and activation, respectively, following intranasal Cm infection. The results showed that the expression of selectins （E-selectin, P-selectin and L-selectin）, and intercellular cell adhesion molecule-1 （ICAM-1） in the lung of C3H mice increased more significantly than in C57BL/6 （B6） mice, the more resistant strain. These results correlated well with the massive neutrophils infiltration in C3H mice. In contrast, CDllb expression on peripheral blood and lung neutrophils in C3H mice exhibited a significant reduction compared with B6 mice during the late phage of infection （day 14）. These findings suggest that the high-level expression of adhesion molecules in C3H mice may enhance neutrophils recruitment to the lung, but the decline of CDllb expression on neutrophils may attenuate neutrophil function. Therefore, CDllb down-regulation on neutrophils may contribute to the failure of C3H mice to control chlamydial lung infection.