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PubMed1996

Leukocyte migration in the leg in response to experimental venous hypertension.

PURPOSE: Leukocyte trapping and activation in the microcirculation of the legs may play an important role in causing skin damage in venous disease. Leukocyte emigration from the microcirculation and subsequent locomotion in response to venous hypertension was studied in a group of 12 normal volunteers using a "skin window" technique. METHODS: Two 0.5-cm square dermal abrasions were made with a dental stone over the gaiter area of the leg and the flexor aspect of the forearm (control), which were covered with moist micropore membranes. The volunteers lay supine for 30 minutes, and then stood supported for 30 minutes to raise the venous pressure in the leg, and then lay supine again for 30 minutes. The experiment was repeated in six volunteers who lay supine for the whole period. The membranes were changed and collected every 15 minutes, fixed in formal saline solution, and dual-stained for monocytes and polymorphonuclear leukocytes. The type and numbers of cells that emigrated and the furthest distance traveled (leading front) by the cells through the membrane were measured. RESULTS: Both in arms and legs, the vast majority of cells that emigrated were neutrophils, with very few monocytes (arm, 93% neutrophils and 7% monocytes; leg, 97% neutrophils and 3% monocytes). In the 30 minutes after venous hypertension, leukocyte migration significantly decreased in the leg (median leukocyte locomotion: basal, 75.3 microns; standing, 73.5 microns; after hypertension, 62.9 microns; p = 0.012, Wilcoxon matched pairs signed rank test), but not in the arm (basal, 86.2 microns; standing, 84.4 microns; after hypertension, 85.5 microns; p = NS) or when the experiment was repeated with the volunteers lying supine for the entire period (basal, 91.5 microns; standing, 89.4 microns; after hypertension, 92.6 microns; p = NS). CONCLUSIONS: Leukocyte migration is decreased immediately after experimental venous hypertension, which may be a result of the release of factors that inhibit migration.

Adult↗
PubMed1987

Inhibitory effect of cetirizine 2HCl on eosinophil migration in vivo.

The effect of a potent antihistamine, cetirizine, was studied on allergic patients and normal subjects by means of an in-vivo 'skin window' technique. All subjects showed significant inhibition of skin-test responses to grass pollen, compound 48/80, histamine and methacholine, after administration of a single dose (10 mg) of cetirizine. Compared to placebo, cetirizine significantly decreased the eosinophils attraction at skin sites challenged with grass pollen and compound 48/80. In allergic patients no change in eosinophil migration pattern was noted with histamine and methacholine skin-tested sites. In normal subjects, compound 48/80 and histamine did not induce eosinophil accumulation and cetirizine did not modify cellular patterns as compared to placebo. These results suggest that cetirizine acts on eosinophil migration by inhibiting the release of mast cell mediators or inhibiting the eosinophilotactic mediators themselves.

Adult↗
PubMed2000

In vivo neutrophil dysfunction in cirrhotic patients with advanced liver disease.

Bacterial infections are frequent, life-threatening complications in cirrhotic patients. This study investigated in vivo neutrophil migration and phagocytic activity in cirrhotic patients with advanced liver disease, in liver transplant recipients, and in healthy volunteers, by use of the skin window technique. Complement receptor type III (CR3) expression was also measured in blood and elicited neutrophils. Neutrophil migration to skin windows and neutrophil in vivo phagocytosis of heat-killed Escherichia coli were significantly decreased in cirrhotic patients compared with healthy controls. Neutrophil migration and phagocytosis were decreased in cirrhotic patients with previous episodes of bacterial infection compared with noninfected patients. Expression of CR3 in circulating neutrophils was significantly higher in cirrhotic patients, whereas it was significantly reduced in elicited neutrophils of cirrhotic patients with previous bacterial infection. These data suggest that deficient neutrophil recruitment to the infection site and impaired phagocytic activity may contribute to bacterial infections in cirrhotic patients with advanced liver disease.

Aged↗
PubMed1992

Skin eosinophilia in patients with allergic and nonallergic asthma and atopic dermatitis.

Skin eosinophilia induced by grass pollen or platelet-activating factor (PAF) was investigated in patients with allergic and nonallergic asthma and atopic dermatitis (AD) and in normal control subjects. Intracutaneous injection of PAF resulted in positive immediate skin reactions in all tested individuals. Since most of the patients with allergic and nonallergic asthma and eczema under investigation were characterized by a peripheral blood eosinophilia, we investigated whether PAF should be capable of mobilizing blood eosinophils to the skin of patients with nonallergic asthma and AD using the Rebuck skin window technique. Our results demonstrated that, in allergic individuals, allergen induced a much stronger skin eosinophilia than PAF and in a larger percentage of the patients (77% versus 45%). There was no correlation between skin eosinophilia induced by PAF and the occurrence of late skin reactions to allergen. Furthermore, the PAF-induced and the allergen-induced skin eosinophilia differed histologically. In nonallergic individuals, neither allergen nor PAF was capable of mobilizing eosinophils in the skin window, in spite of the presence of peripheral blood eosinophilia. Our results indicate that the allergen- and PAF-induced skin eosinophilia are different and that in patients with nonallergic asthma and AD, another chemoattractant than PAF must be responsible for eosinophil mobilization in the target organ.

Adult↗
PubMed1998

Neutrophil functions and IL-8 in psoriatic arthritis and in cutaneous psoriasis.

The aim of this study is to determine some functions of neutrophil in patients affected by psoriatic arthritis and to compare them to those of patients affected by cutaneous psoriasis and to normal controls. We used a model of experimental cutaneous inflammation allowing to separate a cluster of purified and viable PMN cells. Then we analyzed, within the three groups, the IL-8 concentration in serum and in the supernatant obtained from the inflammatory site to gather data on the possible pathogenic role played by this cytokine in psoriatic arthritis. We studied neutrophil functions in patients with cutaneous psoriasis and psoriatic arthritis, in acute phase, in comparison with healthy control subjects. We investigated in vivo neutrophil migration by Senn's skin window technique and measured adhesion assay and superoxide production in circulating and migrating neutrophils after different stimuli. We also measured IL-8 concentration in serum and in the supernatant obtained from the inflammatory site, artificially created through the skin window scrape. Neutrophil migration in vivo was significantly higher in both groups of patients than in controls. In the presence of fMLP, blood cells showed a burst of superoxide release, which was significantly more pronounced in patients when compared to healthy controls. Neutrophils from skin window scrape showed a much higher response to fMLP as compared to blood cells of all subject groups, but no differences were observed between patients and controls. No correlation was found between the three groups in adhesion ability under basal condition or in response to different stimuli by circulating and migrating neutrophils. Our results also show a great increase of IL-8 in the exudate from patients compared to controls. Our study shows that there is no difference in neutrophil functions between patients with psoriatic arthritis and cutaneous psoriasis; moreover we suggest that the source of high IL-8 levels are neutrophils rather than the keratinocytes.

Adult↗
PubMed1991

Platelet-activating factor-induced immediate and late cutaneous reactions.

In atopic subjects, intradermal injection of platelet-activating factor (PAF), 40 and 400 ng, resulted in an immediate edema reaction markedly blocked by cetirizine, 10 mg twice a day. PAF challenge also induced a significant eosinophil accumulation evidenced by a skin window technique at 2, 4, 8 and 24 h. This inflammatory phenomenon was significantly inhibited by cetirizine. In patients with chronic urticaria, PAF, 100 micrograms intradermally, induced immediate and late cutaneous reactions (LCR) also blocked by cetirizine, 10 mg twice a day. These LCR were accompanied by an infiltration of the deep dermis by degranulated eosinophils. The pathophysiological mechanism of the PAF-induced skin reactions is discussed as well as the mechanism of action of cetirizine.

Cetirizine↗
PubMed1989

Delayed maturation of skin window macrophages in myelodysplastic syndromes.

Blood monocyte differentiation to macrophages was examined in nine patients with primary myelodysplastic syndromes using the skin window technique. Emigrated cells were stained cytochemically for acid phosphatase reaction after 1, 2, 4, 7, 9, 12 and 23 h. Compared to age-matched controls, seven patients showed a significant delay in lysosomal enzyme acquisition, which is associated with macrophage differentiation. Our results with this in-vivo assay demonstrate an involvement of the monocyte/macrophage system in primary myelodysplastic syndromes and show that patients often have a disturbance in macrophage differentiation.

Acid Phosphatase↗
PubMed1997

Changes of neutrophil migration without modification of in vitro metabolism and adhesion in Behçet's disease.

OBJECTIVE: Increase of neutrophil chemotaxis in Behçet's disease (BD) has been described, but it is not clear whether there is a correlation with other variables of neutrophil function and whether these modifications correlate with disease activity. METHODS: We studied neutrophil functions in patients with BD in the acute phase in comparison with healthy control subjects and with the same patients during disease remission, with or without therapy. We investigated in vivo neutrophil migration by Senn's skin window technique and measured adhesion assay and superoxide production in circulating and migrating neutrophils after different stimuli. RESULTS: Neutrophil migration in vivo was 101.3 +/- 17.9 x 10(6) polymorphonuclear lymphocytes (PMN)/cm2/24 h in patients with BD in the acute phase and 66.1 +/- 7.8 x 10(6) PMN/cm2/24 h in controls (p < 0.001). No correlation was found between leukocyte counts and neutrophil migration. Neutrophil migration evaluated in the same patients in a phase of disease remission was 58.3 +/- 10.3 x 10(6) PMN/cm2/24 h (p < 0.001 vs acute phase, not significant vs controls). The neutrophils of the exudate were normally primed to response to the chemotactic peptide fMLP. No differences between the 2 groups were found in superoxide production, adhesion under basal conditions, or in response to different stimuli by circulating and migrating neutrophils. CONCLUSION: Abnormally high migration of neutrophils in the active phase of BD is the only consistent neutrophil dysfunction. Since this modification is reversed by therapy, the evaluation of in vivo neutrophil migration may be useful in diagnosing and monitoring disease activity. Blood neutrophils have normal responses to different stimuli, indicating they are not primed by the disease state.

Adult↗
PubMed2005

The human beta-glucan receptor is widely expressed and functionally equivalent to murine Dectin-1 on primary cells.

We identified the C-type-lectin-like receptor, Dectin-1, as the major receptor for fungal beta-glucans on murine macrophages and have demonstrated that it plays a significant role in the cellular response to these carbohydrates. Using two novel, isoform-specific mAb, we show here that human Dectin-1, the beta-glucan receptor (betaGR), is widely expressed and present on all monocyte populations as well as macrophages, DC, neutrophils and eosinophils. This receptor is also expressed on B cells and a subpopulation of T cells, demonstrating that human Dectin-1 is not myeloid restricted. Both major functional betaGR isoforms - betaGR-A and betaGR-B - were expressed by these cell populations in peripheral blood; however, only betaGR-B was significantly expressed on mature monocyte-derived macrophages and immature DC, suggesting cell-specific control of isoform expression. Inflammatory cells, recruited in vivo using a new skin-window technique, demonstrated that Dectin-1 expression was not significantly modulated on macrophages during inflammation, but is decreased on recruited granulocytes. Despite previous reports detailing the involvement of other beta-glucan receptors on mature human macrophages, we have demonstrated that Dectin-1 acted as the major beta-glucan receptor on these cells and contributed to the inflammatory response to these carbohydrates.

Animals↗
PubMed1985

Circadian rhythm of the in vivo migration of neutrophils in psoriatic patients.

Neutrophil chemotaxis in ten subjects with psoriasis was evaluated in vivo by the quantitative skin-window technique using autologous serum and in vitro using a modified Boyden technique. The in vitro chemotaxis values of the psoriatics were significantly lower (P less than 0.01) than those of healthy controls. When measured in vivo, however, we found that, after 9, 12, and 24 h, the values of the migrating polymorphonuclear leukocytes (PMNL) of psoriasis patients were only slightly and non-significantly lower than normal values. Analysing the in vivo chemotaxis data by a chronobiological method, we demonstrated statistically significant circadian rhythms in nine of the ten psoriasis patients. In seven of the patients, we found that the population had a significant rhythm, with the acrophase at -328 degrees. Our in vitro and in vivo values are in agreement with previously published values. However, there was a definite circadian rhythm of migration that we could not demonstrate in normal controls. We consider that our data reveal a significant and important difference between neutrophil chemotaxis in vivo in normal subjects and that in patients with psoriasis.

Adult↗
PubMed1996

Neutrophil migration, oxidative metabolism, and adhesion in elderly and young subjects.

OBJECTIVE: To evaluate neutrophil functions in the elderly. METHODS: We investigated the PMN migration in vivo and PMN superoxide production and adhesion in response to a variety of compounds; PMN have been isolated both from blood and from a skin experimental exudate (obtained by Senn's skin window technique) of 25 normal elderly and of 25 normal young control subjects. RESULTS: No difference was found in PMN migration in vivo (62.9 +/- 21.3 x 10(6) and 65.5 +/- 9.1 x 10(6) PMN/cm2/24 hours in elderly and young subjects respectively), neither were different the adhesion under basal condition and after some stimuli and the superoxide production in basal condition and in response to STZ and PMA in two groups. In elderly subjects superoxide production, in response to fMLP, markedly resulted lower than in young controls both by circulating PMNs (3.6 +/- 2.7 and 9.3 +/- 3.3 nMOLES O2-/10(6) PMN respectively, p < 0.0001) and by exudate PMNs (13.6 +/- 4.3 and 19.4 +/- 6 nMOLES O2-/10(6) PMNs respectively, p < 0.005). CONCLUSION: Many PMN functions in the elderly do not differ from young people, suggesting that the overall defense function of these cells is not affected by aging. The only parameter that we have found to be different between the two groups is the poor superoxide production after fMLP stimulus of PMNs. The stimulus- and function-specificity of this defect in PMNs from elderly subjects indicates the existence of a dysregulation of the signal transduction pathway distal to fMLP receptor and proximal to NADPH oxidase activation.

Adult↗
PubMed1995

Quantitative analysis of angiogenesis and growth of bone: effect of indomethacin exposure in a combined in vitro-in vivo approach.

Nonsteroidal anti-inflammatory agents have been used experimentally and clinically to suppress a variety of physiological events, including angiogenesis and formation of bone. The exact mechanisms by which indomethacin alters skeletal tissue generation are unknown, due in part to methodological limitations. By the use of an organ culture assay and an animal model using intravital microscopy in mice bearing dorsal skinfold chambers, the effect of indomethacin on growth and angiogenesis of neonatal femora was characterized over 16 days. In both assays, femora significantly elongated with time (P < 0.05). The in vitro growth rate was more rapid than in vivo and dependent on the serum concentration, culture medium and age of mice. Although enhancing the serum content promoted cellular proliferation in organ culture, it dose-dependently suppressed femoral elongation, leading at 20% fetal calf serum to growth rates identical to those observed in vivo. Indomethacin supplementation (2 and 10 mg l-1) significantly accelerated longitudinal femoral growth in organ culture (P < 0.05), whereas in vivo indomethacin (2 mg kg-1) did not modulate either angiogenesis or elongation of bone. Our in vitro data propose a central role of serum in the regulation of bone formation. Although indomethacin altered femoral growth in vitro, our findings do not suggest that indomethacin suppresses angiogenesis or growth of bone in vivo. The complexity of physiological events in vivo may be obscuring a detectable effect.

Animals↗
PubMed2005

Effective storage of granulocytes collected by centrifugation leukapheresis from donors stimulated with granulocyte-colony-stimulating factor.

BACKGROUND: Donor stimulation with granulocyte-colony-stimulating factor (G-CSF) has increased the number of neutrophils (PMNs) that can be collected for granulocyte transfusion therapy. Clinical utility, however, has been limited by the inability to store functional PMNs ex vivo. This study was conducted to determine whether granulocyte products from G-CSF-stimulated donors could be effectively stored at reduced temperature (22 degrees C vs. 10 degrees C) with maintenance of functional properties in vitro and in vivo. STUDY DESIGN AND METHODS: Nine normal subjects received G-CSF (600 microg subcutaneously) 12 hours before centrifugation leukapheresis. Granulocyte products were divided and stored for 24 and 48 hours under four conditions: 1) 22 degrees C; 2) 22 degrees C, with supplemental G-CSF (100 ng/mL); 3) 10 degrees C; and 4) 10 degrees C, with supplemental G-CSF. Functional PMN activity during ex vivo storage was assessed in vitro and in vivo by the skin-window technique for granulocytes stored at 10 degrees C for 24 hours. RESULTS: Surface expression of CD11b/CD18, CD14, CD16, CD32, and CD64 was maintained during 48-hour storage at reduced temperature. Inducible respiratory burst activity, bactericidal activity, and fungicidal activity were preserved during storage for 48-hour storage at 10 degrees C. Proinflammatory cytokine production was decreased in product stored at 10 degrees C. Supplemental G-CSF ex vivo did not substantially improve functional activity during storage. After storage at 10 degrees C for 24 hours, in vitro chemotactic potential was maintained, and transfused granulocytes retained capacity to circulate and migrate appropriately in vivo. CONCLUSIONS: Granulocyte product collected by centrifugation leukapheresis from G-CSF-stimulated donors can be effectively stored at subphysiologic temperature for 24 hours with preservation of functional activity. Storage at 10 degrees C appears to be slightly superior to storage at 22 degrees C.

Adult↗
PubMed1977

Levels of carbenicillin, ticarcillin, cephalothin, cefazolin, cefamandole, gentamicin, tobramycin, and amikacin in human serum and interstitial fluid.

The ability of eight antibiotics (carbenicillin, ticarcillin, cefamandole, cephalothin, cefazolin, gentamicin, tobramycin, and amikacin) to enter human interstitial fluid was evaluated by the skin window technique. All of the antibiotics tested, except cefazolin, which has the highest percentage of protein binding, diffused into the interstitial fluid quite well. This study confirms our previous observation on the effect of high-percentage protein binding on diffusion of a drug into the minimally inflamed extravascular space.

Adult↗
PubMed1984

Effect of high-dose methylprednisolone therapy on phagocyte function in systemic lupus erythematosus.

Circulating phagocytes play a major role in the defence of the host against microbial infection. In an attempt to identify the reason for the unusual susceptibility to infection of patients with systemic lupus erythematosus (SLE) various parameters of phagocytic cell function were assessed kinetically in whole blood, and the accumulation of cells in areas of inflammation was studied in vivo with the skin window technique. The effect on these parameters of conventional therapy with glucocorticoids and pulse therapy with large doses of methylprednisolone were examined. Patients on conventional doses of steroids had no abnormality of phagocyte function that might have predisposed to infection, apart from a reduced accumulation of monocytes in areas of inflammation and decreased lactoferrin secretion. Pulse therapy with methylprednisolone considerably delayed the secretion of lactoferrin and the adherence of neutrophils in most of the patients, as well as impairing bacterial killing and digestion.

Adult↗
PubMed1986

Prognostic significant of functional defects of granulocytes in myeloproliferative disease.

Patients suffering from myeloproliferative diseases are frequently susceptible to infection. We studied granulocyte function and cytogenetics in 32 patients with different myeloproliferative syndromes. In vivo granulocyte function was studied in 10 patients using the skin window technique. Our data demonstrate a deficit in chemotaxis (p less than 0.001) which does not seem to correlate with any specific chromosomal anomaly. Patients showing a deficit in myeloperoxidase activity, phagocytosis, O2- production or NBT reduction also had a much lower median survival with respect to other patients treated with the same protocol but without these deficits (p less than 0.01). By conducting granulocyte function studies at diagnosis one may therefore isolate those patients with an increased risk of contracting infections and a decreased probability of complete remission.

Adult↗
PubMed1968

Leukokinetic studies. 13. A non-steady-state kinetic evaluation of the mechanism of cortisone-induced granulocytosis.

The mechanism by which adrenocortical steroids induce granulocytosis in man has been investigated using granulocytes labeled with radioactive diisopropylfluorophosphate. After an intravenous injection of 200 mg of cortisol was given to five normal subjects, the mean value for the total blood granulocyte pool increased from 79 to 138 x 10(7) cells per kg of body weight and reflected an increase in the size of both the circulating granulocyte pool and the marginal granulocyte pool. When granulocytes in the circulation were labeled with diisopropylfluorophosphate and granulocytosis was induced later by the intravenous administration of cortisol, the rate of decline of granulocyte specific activity was increased, indicating that the blood pool was being diluted at an accelerated rate by unlabeled cells entering from the bone marrow. The rate of egress of granulocytes from the blood pool to an inflammatory exudate was studied by the "skin window" technique. After the administration of cortisol, there was a mean reduction in the cellularity of induced inflammatory exudates of 75%. However, this reduction in cellularity varied considerably from subject to subject (45-98%). From these studies we can infer that steroids induce an absolute granulocytosis by decreasing the rate of egress of cells from the total blood granulocyte pool as well as by increasing the influx of cells from the bone marrow. By model simulation studies of the non-steady state induced by cortisol injection, it has been possible to quantitate these rate changes. In the present studies cortisol injection resulted in a mean decrease in blood granulocyte egress of 74% (1-99%) and a mean increase in cell inflow of 450% (300-750%).

Adult↗