A method for multiple sequential analyses of macrophage functions using a small single cell sample

Microbial pathogens such as bacillus Calmette-Guérin (BCG) induce the activation of macrophages. Activated macrophages can be characterized by the increased production of reactive oxygen and nitrogen metabolites, generated via NADPH oxidase and inducible nitric oxide synthase, respectively, and by the increased expression of major histocompatibility complex class II molecules (MHC II). Multiple microassays have been developed to measure these parameters. Usually each assay requires 2-5 x 10(5) cells per well. In some experimental conditions the number of cells is the limiting factor for the phenotypic characterization of macrophages. Here we describe a method whereby this limitation can be circumvented. Using a single 96-well microassay and a very small number of peritoneal cells obtained from C3H/HePas mice, containing as little as <=2 x 10(5) macrophages per well, we determined sequentially the oxidative burst (H2O2), nitric oxide production and MHC II (IAk) expression of BCG-activated macrophages. More specifically, with 100 æl of cell suspension it was possible to quantify H2O2 release and nitric oxide production after 1 and 48 h, respectively, and IAk expression after 48 h of cell culture. In addition, this microassay is easy to perform, highly reproducible and more economical

A method for multiple sequential analyses of macrophage functions using a small single cell sample.

Microbial pathogens such as bacillus Calmette-Guérin (BCG) induce the activation of macrophages. Activated macrophages can be characterized by the increased production of reactive oxygen and nitrogen metabolites, generated via NADPH oxidase and inducible nitric oxide synthase, respectively, and by the increased expression of major histocompatibility complex class II molecules (MHC II). Multiple microassays have been developed to measure these parameters. Usually each assay requires 2-5 x 10(5) cells per well. In some experimental conditions the number of cells is the limiting factor for the phenotypic characterization of macrophages. Here we describe a method whereby this limitation can be circumvented. Using a single 96-well microassay and a very small number of peritoneal cells obtained from C3H/HePas mice, containing as little as <=2 x 10(5) macrophages per well, we determined sequentially the oxidative burst (H2O2), nitric oxide production and MHC II (IAk) expression of BCG-activated macrophages. More specifically, with 100 micro l of cell suspension it was possible to quantify H2O2 release and nitric oxide production after 1 and 48 h, respectively, and IAk expression after 48 h of cell culture. In addition, this microassay is easy to perform, highly reproducible and more economical.

Red blood cells stimulate fibroblast-mediated contraction of three dimensional collagen gels in co-culture.

OBJECTIVE AND DESIGN: Following injury, red blood cells (RBC) may interact with extracellular matrix (ECM). In the present study we hypothesised that RBC, and soluble factors from RBC, might mediate remodelling of ECM by affecting fibroblast-mediated contraction of three dimensional collagen gels. MATERIALS AND METHODS: Human lung fibroblasts (HFL-1), were cultured together with isolated RBC, conditioned medium from RBC (RBC-CM) and hemolysed RBC in type I collagen gels. Gel contraction was determined by an image analyser. RESULTS: Both RBC, RBC-CM and hemolysed RBC stimulated gel contraction by fibroblasts (P < 0.001), compared to fibroblasts alone. The RBC-CM stimulated (P < 0.01) gel contraction in a time and concentration dependent manner. A similar effect was observed when supernatant from hemolysed RBC was tested. The production of fibronectin was increased (P < 0.01) in the co-culture system, compared to fibroblasts cultured alone. CONCLUSIONS: The present study shows that RBC can interact with mesenchymal cells in vitro. The ability of RBC to modulate fibroblast-mediated contraction in vitro, might therefore be an important mechanism regulating repair processes after injury.

Effects of birch pollen and traffic particulate matter on Th2 cytokines, immunoglobulin E levels and bronchial hyper-responsiveness in mice.

BACKGROUND: Health effects due to air pollution arising from motor vehicles are a major public and political concern world-wide. Epidemiological studies have shown that the manifestations of asthma are increased by air pollution in already affected individuals. OBJECTIVE: To investigate the potential role of air-polluted tunnel dust (traffic particulate matter, TPM) or pure carbon core particles in the initiation and persistence of experimental allergic inflammation. METHODS: BP2 mice were immunized with birch pollen alone (group B) or pollen together with TPM (group A), or with birch pollen and Al(OH)3 (group C), or with birch pollen and carbon core particles (group D). Before methacholine challenge they were challenged intranasally and thereafter bronchial hyper-reactivity (BHR) was evaluated in a whole-body plethysmograph. Levels of Th2 cytokines, fibronectin and lactate dehydrogenase (LDH) were determined, and differential counts were performed in the bronchoalveolar lavage (BAL) fluid. Sera were collected for determination of antibody titres and cytokine levels. RESULTS: Specific IgE titres, BHR, the number of recruited eosinophils and levels of fibronectin and LDH in BAL were increased in mice immunized and challenged with a mixture of birch pollen and TPM. However, mice immunized with birch pollen alone and challenged intranasally with pollen or a mixture of pollen and TPM demonstrated the highest levels of IL-4 and IL-5. CONCLUSION: This study highlights the importance of the exposure to a combination of particulate matters and pollen allergens, in the induction of allergic disease in the airways, and we have demonstrated that polluted tunnel dust has an effect on both the inflammatory and immunological components of experimental allergy. Immunization and challenge with carbon core particles together with birch pollen increased neither the BHR nor the specific IgE production significantly. Our results therefore strongly suggest that it is most likely to be the organic phase bound to the carbon core of the diesel exhaust particles that might have an important adjuvant effect in the induction of experimental allergy.

Impaired monocyte CD11b expression in interstitial inflammation in hemodialysis patients.

BACKGROUND: It is not known to what extent intravascular phenotypic alterations in adhesion molecule expression induced by hemodialysis influence the recruitment of monocytes and their ability to up-regulate CD11b at the local site of inflammation in the interstitium. Using a skin suction chamber technique, we addressed these issues in eight hemodialysis patients and in eight healthy subjects. METHODS: Two skin blisters were raised on the forearm of each individual and blister exudate collected. The blisters were then stimulated with autologous serum (active blister, intense inflammation) or buffer (control blister, intermediate inflammation), respectively. Thereafter the patients were treated with Cuprophan hemodialysis for four hours. After 10 hours, the exudate was aspirated from each chamber in all subjects. Monocyte count and expression of CD11b were analyzed in serum and blister fluid by flow cytometry. Then, monocytes from healthy blood donors were incubated in blister fluid from patients and healthy subjects in order to determine the local chemotactic activity in terms of CD11b up-regulation. Monocyte chemotactic protein-1 (MCP-1), a marker of systemic monocyte chemotactic activity, was also analyzed in serum at 0 and 10 hours in all individuals. RESULTS: The number of monocytes at the site of inflammation in the interstitium in hemodialysis patients correlated with the expression of CD11b on transmigrated cells (r = 0.78, P < 0.001). Monocytes collected in the active blister fluid of dialysis patients expressed equal levels of CD11b as cells collected from healthy subjects. By contrast, monocytes collected from the control blisters of patients expressed lower levels of CD11b than cells from healthy subjects (P < 0.01), despite equal interstitial biological activity of CD11b-mobilizing factors in blister fluid from patients and healthy subjects and the fact that patients had higher systemic chemotactic activity in terms of MCP-1 concentration in serum (P < 0.001). CONCLUSION: Monocytes from hemodialysis patients have the capacity to mobilize CD11b to the same extent as cells from healthy individuals at the inflammatory spot, but more intense stimuli are required for such actions, probably because of a transient refractoriness.

The impact of eotaxin- and IL-5-induced adhesion and transmigration on eosinophil activity markers.

Eosinophils accumulate at sites of allergic inflammation, and play important roles in asthma/allergic disorders. The mechanism of eosinophil recruitment into tissues is not fully understood. In this study, we evaluated whether adhesion and/or transmigration, in the presence of IL-5 and eotaxin, alter the expression of CD9, CD11b, the beta1alpha4-integrin, and the EG2-epitope on intracellular ECP. We also investigated whether CD9 is involved in the adhesion process. With flow cytometry the surface expression of CD9, CD11b and the beta1alpha4-integrin, and the intracellular expression of EG2, were analyzed before, and after transmigration/adhesion to fibronectin. To evaluate the eventual role of CD9 in adhesion, eosinophils were preincubated with monoclonal antibodies to CD9. We observed decreased expression of CD9, and increased expression of CD11b on eosinophils, after adhesion and transmigration. The transmigration did not change the expression of the beta1alpha4-integrin or EG2, whereas the adhesion resulted in a decreased EG2 expression. Antibodies to CD9 decreased the adhesion property of eosinophils. The eosinophils are activated after both adhesion and transmigration by means of decreased CD9 and increased CD11b expression. The expression of the EG2-epitope on intracellular ECP was decreased when eosinophils adhered to fibronectin, probably due to degranulation. Our results also indicate that CD9 is involved in the adhesion of eosinophils to fibronectin.

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.

Allergen-induced accumulation of eosinophils and lymphocytes in skin chambers is associated with increased levels of interleukin-4 and sVCAM-1.

BACKGROUND: The aim of the study was to characterize the kinetic accumulation of various inflammatory mediators in allergen-challenged skin chambers applied on patients with pollen-related allergic rhinitis/mild asthma. METHODS: Skin blisters were induced on the forearms and challenged with allergen or phosphate-buffered saline (PBS). Peripheral blood was drawn before and 8 h after challenge for analysis of differential cell counts, sVCAM-1, and alpha2-macroglobulin. Chamber fluids, collected at 1, 4, and 8 h after allergen application, were analyzed for differential cell counts, histamine, interleukin (IL)-4, sVCAM-1, and alpha2-macroglobulin. RESULTS: The number of recruited leukocytes was equal in allergen and PBS chambers; however, the numbers of eosinophils and lymphocytes were significantly (P< or =0.05) elevated in allergen-challenged chambers at 8 h. Compared to PBS chambers, allergen chambers contained significantly (P<0.01-0.05) higher levels of histamine (at 1 and 4 h), IL-4 (at 4 and 8 h), alpha2-macroglobulin (at 1 and 8 h), and sVCAM-1 (at 1 and 8 h). In contrast to alpha2-macroglobulin, levels of sVCAM-1 in peripheral blood were significantly (P<0.05) increased at 8 h. CONCLUSIONS: Increased levels of sVCAM-1 and IL-4 in allergen-challenged chambers, in parallel with increased recruitment of eosinophils and lymphocytes, points to the participation of IL-4 and VCAM-1 in the development of the late-phase reaction. Increased levels of sVCAM-1 in allergen-challenged chambers probably reflects a combination of leakage and local production.

The effect of in vitro activation and platelet interaction on the CD9 distribution and adhesion properties of human eosinophils.

OBJECTIVE AND DESIGN: The redistribution of CD9 in peripheral blood eosinophils was investigated with respect to the interaction with platelets during in vitro activation, and whether this interaction exerts influence on eosinophil adhesion properties. MATERIALS AND METHODS: Flow cytometry was used to investigate the CD9 expression in purified eosinophils or eosinophils from different whole blood preparations, with or without platelets present. To confirm an eosinophil/platelet interaction fluorescence microscopy was used, and to demonstrate release/shedding of CD9 molecules a biosensor technique was performed. RESULTS: Our results show that both intracellular and surface expression of CD9 decrease upon in vitro activation in the absence of platelets, a phenomenon probably caused by release/shedding of soluble forms of CD9 and not due to intracellular degradation. Increased expression of CD9 on eosinophils, stimulated in the presence of platelets, is partly a result of interacting platelets, judged by the increase in platelet specific marker CD61. In our adhesion assay a significant increase in eosinophil adhesion properties to fibronectin was obtained when eosinophils were PMA stimulated and interacting with platelets, as compared to activated eosinophils without platelets. CONCLUSIONS: Our findings, that CD9 expression on eosinophils is dynamically regulated, support our previous suggestion that CD9 may be a useful activity marker and that platelet interaction acts on eosinophil adhesion.

Ropivacaine inhibits leukocyte rolling, adhesion and CD11b/CD18 expression.

Ropivacaine, a new local anesthetic, is currently being investigated for the treatment of ulcerative colitis, with promising results so far. The aim of this study was to examine anti-inflammatory properties of ropivacaine with regard to its effects on vascular permeability and inflammatory leukocyte behavior in vivo. The effects on leukocyte rolling, firm adhesion and vascular permeability were examined in the hamster cheek pouch microvasculature via intravital microscopy, and the effects on leukocyte adhesion molecules were examined in vitro by means of flow cytometry. In large venules, leukocyte adhesion induced by topical leukotriene B4 (LTB4) was almost completely inhibited during the combined application of ropivacaine and LTB4. The spontaneous rolling leukocyte flux was reduced by 72%, the rolling leukocyte fraction by 47% and the total leukocyte flux, which reflects blood flow, by 47%. In postcapillary venules, ropivacaine abolished rolling and LTB4-induced firm adhesion of leukocytes. LTB4 challenge also resulted in increased plasma exudation that was almost completely inhibited by ropivacaine. Moreover, ropivacaine inhibited the tumor necrosis factor alpha-induced up-regulation of CD11b/CD18 and L-selectin shedding by human leukocytes in vitro. Our results suggest that ropivacaine exerts anti-inflammatory activity, and this appears to be mediated to a significant extent by inhibition of both leukocyte rolling and adhesion.