Heparin protects against skin flap necrosis: relationship to neutrophil recruitment and anti-coagulant activity.

OBJECTIVE AND DESIGN: Heparin has been shown to improve survival of surgical skin flaps. However, it is not known whether the protective effect of heparin is related to its anticoagulative or anti-inflammatory effects. METHODS: Surgical flaps were raised in the dorsal skin of Sprague-Dawley rats. Neutrophil recruitment was determined by measuring the tissue content of myeloperoxidase (MPO) and clotting time was estimated by assessment of activated partial thromboplastin time (APTT) in plasma. RESULTS: Administration of heparin (150 U/kg) significantly increased skin flap survival from 44% in vehicle-treated controls to 91%. This heparin treatment increased APTT by 4.5 fold. However, administration of 150 U/kg of heparin had no effect on skin flap neutrophil recruitment. In contrast, we found that the polysaccharide fucoidan reduced MPO and also improved skin flap survival. CONCLUSIONS: In conclusion, we demonstrate that protective effect of clinically relevant doses of heparin correlates with its ability to prolong clotting time and not to inhibition of neutrophil accumulation in the healing of skin flaps.

Increased tissue survival in experimental skin flaps in mast cell-deficient rats.

AIM: The role of mast cells and their principal mediator, histamine, in surgical skin flap survival was investigated using mast cell-deficient (Ws/Ws); their congenic littermates wild-type (+/+), and Wistar rats. METHODS: A standardized dorsal skin flap was raised and sutured back into position, and 6 days later the percentage of flap survival was assessed. Moreover, endogenous histamine concentration in the dorsal skin during the surgical preparation was determined using in vivo microdialysis technique together with high performance liquid chromatography-fluorometry. Accumulation of skin flap myeloperoxidase (MPO) (reflecting leucocyte recruitment) was determined spectrophotometrically. RESULTS: The experimental skin flaps in genetically mast cell-deficient rats exhibited increased tissue survival and showed little accumulation of MPO and rather low and stable level of histamine output in comparison with skin flaps in the wild-type (+/+) littermates or normal Wistar rats. Antihistamine treatment inhibited but did not prevent leucocyte recruitment in the skin flaps post-surgery in +/+ and Wistar rats. CONCLUSION: It is suggested that mast cell derived histamine plays an important role in leucocyte recruitment in skin flaps. However, mast cell-independent factors should be taken into consideration and needs further investigation as even in mast cell-deficient animals there was some accumulation of leucocytes and tissue necrosis in the skin flaps post-surgery.

Differential protein expression in rat trigeminal ganglia during inflammation.

A proteomics approach was used to investigate global protein changes in rat sensory ganglia exposed to pro-inflammatory stimuli. Inflammation was provoked in vivo by injecting selected facial areas with Freunds Complete Adjuvant (FCA), or by stimulating freshly isolated trigeminal ganglia ex vivo with pro-inflammatory mediators (interleukin-1 beta, tumor necrosis factor-alpha, interferon-gamma). Following two-dimensional gel electrophoresis, silver staining and mass spectrometry, a protein reference map and database was generated for rat trigeminal ganglia, which to our knowledge is the first to be reported. Sixty-seven out of 85 selected protein spots were successfully identified using matrix-assisted laser desorption/ionization mass spectrometry. This reference map was used to monitor changes in the ganglia proteome induced during inflammation in vivo and ex vivo. In vivo we found that FCA treatment specifically induced differential protein expression of two unidentified protein spots and, to a lower extent, of beta-tubulin. Image analysis of ganglia treated ex vivo with the cocktail of cytokines indicated that some of the changes in the protein population were also observed in vivo after FCA treatment. If the cytokine stimulation was performed in the presence of acetaminophen (paracetamol), the drug seemed to reverse the effects of cytokine treatment for at least some protein spots, restoring the same protein pattern observed in control samples.

Role of CD18-dependent neutrophil recruitment in skin and intestinal wound healing.

CD11/CD18 is an important adhesion molecule mediating recruitment of leukocytes, which, in turn, may cause postoperative injury in the skin and gastrointestinal tract. The objective of the present study was to investigate the effects of inhibiting the function of CD18 on surgery-induced dermal and intestinal infiltration of neutrophils and on the healing of surgical skin flaps and colonic anastomosis. A flap in the dorsal skin or an end-to-end colonic anastomosis were created in Sprague-Dawley rats. Skin necrosis and anastomotic breaking strength were analyzed 6 and 3 days after surgery, respectively. Tissue myeloperoxidase (MPO) was used as a marker of neutrophil recruitment. Administration of a monoclonal antibody directed against rat CD18 (WT.3, 2 mg/kg) significantly decreased dermal and anastomotic MPO activity by more than 80%. Passive immunization against CD18 significantly improved flap survival, i.e. the survival was 80% in the anti-CD18 antibody group as compared to 38% in the control group. In contrast, this passive immunization against CD18 had no effect on the reconstitution of the integrity of the colonic anastomosis, i.e. the anastomotic breaking strength was 1.3 +/- 0.1 and 1.3 +/- 0.3 N in the control and anti-CD18 antibody group, respectively. These findings suggest that specific inhibition of CD18 function and reduced neutrophil recruitment may improve the survival of experimental skin flaps and, thus, may represent a potential target for therapeutic intervention. In contrast, we also found that blocking CD18-dependent neutrophil infiltration in the intestine had no effect on breaking strength of colonic anastomosis. Thus, neutrophils may influence the wound-healing process differently in specific organs and this needs to be considered when applying an anti-inflammatory treatment regime in order to improve tissue healing.

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.

Low molecular weight heparin as adjuvant therapy in active ulcerative colitis.

BACKGROUND: Heparin given intravenously has shown beneficial effects in the treatment of refractory ulcerative colitis in open trials. Low molecular weight heparin (LMWH) offers advantages in the method of administration but have not been evaluated in inflammatory bowel disease conditions. AIM: To assess the tolerability and safety of subcutaneous self-administered LMWH in outpatients with refractory ulcerative colitis and to evaluate any potential adjuvant therapeutic effect. PATIENTS AND METHODS: Twelve patients with mild to moderately active ulcerative colitis were included in the trial. The patients had either responded poorly to treatment with conventional therapy, including oral and/or rectal glucocorticosteroids, or had experienced a rapid relapse during or shortly after GCS therapy. Dalteparin sodium 5000 units s.c. injection was administered twice daily for 12 weeks. Patients were monitored for possible adverse events and changes in clinical symptoms, and endoscopic and histological scores were analysed. Leucocyte scanning was performed at inclusion and at the end of the study. RESULTS: Tolerability and compliance were excellent and no serious adverse events occurred. Eleven patients improved symptomatically and six (50%) attained complete remission after 12 weeks of treatment. Endoscopic, scintigraphic and histological scores were found to be significantly improved. CONCLUSION: Self-administered LMWH given s.c. may be a safe adjuvant therapy for patients with active, glucocorticosteroids-refractory ulcerative colitis. A controlled trial should be undertaken to confirm the positive effects found in this study.

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.

Effects of polysaccharide fucoidin on cerebrospinal fluid interleukin-1 and tumor necrosis factor alpha in pneumococcal meningitis in the rabbit.

The inflammatory response in bacterial meningitis is mediated by cytokines, such as tumor necrosis factor alpha (TNF-alpha) and interleukin-1 (IL-1), which are produced in the subarachnoid space by different cells, e.g., leukocytes, astrocytes, and microglia. The recruitment of leukocytes into the cerebrospinal fluid (CSF) has been shown to contribute to the neurological damage in this disease, a process which could be enhanced by treatment with antibiotics. In this study, we have used a rabbit meningitis model for two sets of experiments with intracisternal (i.c.) injections of Streptococcus pneumoniae. First, pneumococcal cell wall (PCW) components were injected i.c., inducing an inflammatory response with pleocytosis and increased levels of CSF TNF-alpha) and IL-1 at 6 and 12 h after PCW injection. Treatment with fucoidin, known to inhibit leukocyte rolling, abolished pleocytosis and inhibited the release of TNF-alpha and IL-1. In the second experiment, live pneumococcal bacteria were injected i.c. and treatment with one dose of ampicillin (40 mg/kg of body weight intravenously) was given 16 h after induction of meningitis, causing a sevenfold increase in CSF leukocytes over a 4-h period. CSF IL-1 levels at 16 h were high but did not increase further at 20 h. Also, CSF TNF-alpha levels were high at 16 h and tended to increase at 20 h. Fucoidin treatment prevented the antibiotic-induced increase of CSF leukocytes but had no effect on the TNF-alpha and IL-1 levels. Taken together, fucoidin reduced CSF TNF-alpha and IL-1 levels in acute bacterial meningitis induced by PCW fragments but had no effect later in the course of the disease, when live bacteria were used and an inflammatory increase was caused by a dose of antibiotics.

Effects of calcitonin gene-related peptide on tissue survival, blood flow and neutrophil recruitment in experimental skin flaps.

Local administration of calcitonin gene-related peptide (CGRP) has been shown to improve tissue survival in surgical skin flaps. Moreover, topical CGRP has been demonstrated to exert anti-inflammatory effects in different animal models of skin inflammation. The aim of the present study was to establish whether systemic treatment with low doses of CGRP may improve survival and reduce neutrophil accumulation in surgical skin flaps. Using a well-established dorsal skin-flap model in the rat, we found that intraperitoneal (i.p.) pretreatment with low doses of CGRP dose-dependently increased flap survival. Thus, in untreated animals flap survival at day 7 after surgery was 42%, as compared to 44%, 60%, 69% and 73% survival after a single preoperative i.p. injection of 10(-15), 10(-12), 10(-9) and 10(-6) mol CGRP, respectively (P < 0.05 versus control for the three highest doses). The three effective doses had no detectable effects on either flap blood flow (laser Doppler) or mean arterial blood pressure. On the other hand, 5 x 10(-9) mol CGRP i.p. significantly reduced the marked surgery-induced accumulation of flap myeloperoxidase (a marker for neutrophil recruitment) without affecting the circulating neutrophil count. Taken together, our findings demonstrate that low systemic doses of CGRP can cause a major improvement in skin-flap survival in the rat, possibly via inhibition of surgically induced neutrophil recruitment.

Using mothers' and newborns' clinical anthropometric data to analyse birth histories.

The aim of the study was to measure the effect of mothers' and babies' various anthropometric parameters on the deviation from the normal course of pregnancy and labour. For this purpose the clinical anthropometric parameters of 532 parturients (primipara) and their newborns, and some additional indices formed from these data were correlated with the sum in points of all individual deviations from the normal course of anamnesis, pregnancy and childbirth as independent risk factors ("birth index" BI). The analysis showed that mothers' and babies' anthropometric data are essential co-factors in the formation of the total risk for mothers' and babies' health. Our investigation has demonstrated that a two-dimensional classification formed from height and parturient's complex body build index (PCBBI) (3 x 3 SD classes with appropriate statistical data-processing) can form a common methodological basis for using anthropometric characteristics in evaluation of obstetric data. As in the future analogous classifications could be used on obstetric material by different authors, the content of all corresponding classes--the mean values of newborns' birthweight and the birth index--would also be statistically comparable.

The polysaccharide fucoidin inhibits the antibiotic-induced inflammatory cascade in experimental pneumococcal meningitis.

There is evidence that the treatment of bacterial meningitis with antibiotics liberates harmful bacterial products in the subarachnoid space (SAS). This enhances meningeal inflammation and in particular the recruitment of leukocytes into the cerebrospinal fluid (CSF), which has been shown to be more harmful than beneficial in this disease. In this study, we used a rabbit meningitis model based on intracisternal injection of live Streptococcus pneumoniae. Ampicillin (40 mg/kg of body weight given intravenously [i.v.] 16 h after induction of meningitis) caused a fivefold increase in CSF leukocytes over a 4-h period. Inhibition of leukocyte rolling by treatment with the polysaccharide fucoidin (10 mg/kg, i.v.) prevented the enhanced leukocyte extravasation into the SAS and attenuated the leakage of plasma proteins over the blood-brain barrier. These results suggest that certain polysaccharides that block leukocyte rolling have the potential to reduce leukocyte-dependent central nervous system damage in bacterial meningitis.

An approach for studies of mediator-induced leukocyte rolling in the undisturbed microcirculation of the rat mesentery.

1. Although intravital microscopy is the method of choice for observation of inflammatory leukocyte rolling and adhesion in small venules in vivo, a problem with this technique is that surgical exposure of suitable tissues per se triggers the rolling mechanism. In this study, we describe an approach to investigate induction of rolling in undisturbed microvessels. For this purpose, intravital microscopic observation of leukocyte rolling and adhesion in the rat mesentery was combined with histological determination of the intravascular concentrations of polymorphonuclear and mononuclear leukocytes (PMNL and MNL). 2. By relating the histologically determined number of intravascular leukocytes to either microvessel volume or to the erythrocyte concentration, the baseline MNL and PMNL content was found to be 3-6 fold higher in venules than in systemic blood. This increase in microvessel leukocyte concentration did not seem to be related to leukocyte-endothelium interactions, because the leukocyte concentration was similarly elevated in arterioles where rolling and adhesion did not take place. 3. Preparation of the rat mesentery for intravital microscopy time-dependently increased the venular PMNL concentration to over 100 fold the systemic PMNL concentration 45 min after exteriorization of the small intestine. The MNLs were much less responsive to the preparative manipulation. By treatment with the polysaccharide fucoidin (inhibits rolling but not firm adhesion per se), or by use of intravital microscopy immediately before tissue fixation, approximately 90% of the accumulated venular PMNLs were found to represent rolling cells. 4. Intraperitoneal injection of 10(-3) M histamine increased the venular PMNL (but not the MNL) concentration to almost 50 fold the systemic PMNL value. The histamine response did not vary with venular diameter, and the relative contribution of rolling vs firmly adherent cells to the PMNL, accumulation was again approximately 90%. Intraperitoneal injection of leukotriene C4, but not prostaglandin E2, caused a significant increase in venular PMNL concentration. 5. Systemic treatment with the anti-P-selectin monoclonal antibody PB1.3 had no effect on the histamine-induced venular PMNL accumulation (i.e. rolling) in female Wistar or male Sprague-Dawley rats. On the other hand, identical treatment with PB1.3 very effectively inhibited the histamine-induced PMNL response in the mesentery of rabbits. 6. In conclusion, we have shown that a histologically determined increase in leukocyte concentration in rat mesenteric venules may be used as an index of mediator-induced leukocyte rolling if the relative contribution of rolling and firm leukocyte adhesion is first determined, for example by the means described in this study. This relatively simple approach may be very useful for studying various aspects of leukocyte rolling when the 'spontaneous' rolling triggered by preparation of tissues for intravital microscopy is undesirable.

Characteristics of histamine-induced leukocyte rolling in the undisturbed microcirculation of the rat mesentery.

1. The main objective of this study was to analyse the role and mode of action of the mast cell mediator histamine in leukocyte-endothelium interactions in small venules in vivo. For this purpose, we used a histological approach (combined with intravital microscopy) that allows studies of rapid mediator-induced venular leukocyte accumulation, reflecting leukocyte rolling, in the undisturbed microcirculation of the rat mesentery where rolling is normally absent. 2. We first examined the relative importance of histamine and 5-hydroxytryptamine (5-HT) in acute mast cell-dependent leukocyte recruitment. The mast cell secretagogue compound 48/80 (i.p. for 15 min) induced a marked venular accumulation of polymorphonuclear leukocytes (PMNL) which was almost abolished by combined histamine1 (H1)- and histamine2 (H2)-receptor blockade. In contrast, the 5-HT-receptor antagonist methysergide was inactive in this regard. Moreover, exogenous 5-HT was less active than exogenous histamine in evoking venular PMNL accumulation (histamine response dose-dependent; 5-HT response bell shaped). Prostaglandin D2 did not cause PMNL accumulation. 3. The venular PMNL response to exogenous histamine peaked between 15 min and 1 h, was still significantly elevated at 2 h, and then returned to prechallenge values after 3 h. At all time points, the histamine-induced PMNL accumulation was nearly abolished by i.v. treatment with the polysaccharide fucoidin (which blocks rolling but not firm adhesion per se), suggesting that the PMNL response to histamine was due to rolling rather than firm adhesion over the entire 3 h period. At no time point did histamine trigger accumulation of mononuclear leukocytes (MNL). 4. To examine the role of histamine-receptors in the histamine-induced PMNL accumulation (i.e. rolling), the animals were pretreated with diphenhydramine (H1-receptor antagonist), cimetidine, or ranitidine (H2-receptor antagonists). Diphenhydramine alone inhibited the venular PMNL response to histamine by 52%, while both H2-receptor antagonists were completely inactive. However, the combination of cimetidine and diphenhydramine reduced the histamine-induced PMNL rolling by 82%. Furthermore, in contrast to an H3-receptor agonist, challenge with either the H1-receptor agonist 2-thiazolylethylamine or two different H2-receptor agonists (impromidine, dimaprit) was sufficient to provoke significant venular PMNL accumulation. 5. Treatment with the nitric oxide-synthase inhibitor L-NAME did not affect the histamine-induced PMNL rolling. On the other hand, 3 h pretreatment with dexamethasone reduced the PMNL response to histamine by 73%, and flow cytometric analysis showed that the dexamethasone treatment almost completely inhibited binding of soluble P-selectin to rat isolated PMNLs. 6. We conclude that initial leukocyte recruitment after mast cell activation in the rat mesentery is critically dependent on histamine release. The cellular response to histamine was specifically due to PMNL rolling, involved activation of both H1- and H2-receptors, and lasted for 2 3 h. Moreover, the histamine-induced PMNL rolling was not dependent on nitric oxide synthesis, but was sensitive to glucocorticoid treatment, possibly via inhibition of expression or function of leukocytic P-selectin ligand(s).

In vivo rolling and endothelial selectin binding of mononuclear leukocytes is distinct from that of polymorphonuclear cells.

In inflammation, rolling of leukocytes along the microvascular endothelium is a precondition for subsequent integrin-mediated firm adhesion and extravasation. Rolling characteristics of polymorphonuclear leukocytes (PMNL) and mononuclear leukocytes (MNL) in small venules (15-25 microns) of the rat mesentery were studied by intravital fluorescence microscopy under basal conditions and after intravenous treatment with an anti-rat neutrophil serum (ANS). The baseline rolling fraction of the venular total leukocyte flux was 36 +/- 15% (mean +/- SD). The PMNL fraction of the systemic leukocyte count was 27 +/- 9%. Treatment with ANS resulted in total depletion of circulating PMNL and reduced the leukocyte rolling fraction to 12 +/- 5%, in this situation represented only by MNL. In rats treated intraperitoneally with interleukin (IL)-1 beta for 4 h, the leukocyte rolling fraction was 53 +/- 13% and was reduced to 33 +/- 11% after ANS treatment. These data indicated that most, if not all, circulating PMNL rolled along the venular endothelial lining in the rat mesentery prepared for intravital microscopy, whereas MNL rolling was minor (approximately 10%) under the same basal condition. In cytokine-activated tissue, on the other hand, the number of rolling MNL was greatly increased. While PMNL rolling is known to be entirely selectin dependent, the increased MNL rolling after IL-1 stimulation was likely mediated by alpha 4 integrins, inasmuch as the rolling fraction of isolated peripheral blood lymphocytes injected into the microcirculation of the cytokine-stimulated mesentery was reduced from 31 +/- 14% to 6 +/- 2% by pretreatment of the cells with a monoclonal antibody against the rat integrin alpha 4 chain. In accordance with the in vivo rolling characteristics of the two cell populations, binding of soluble P- or E-selectin (selectin/IgG chimeras) was less intense for blood lymphocytes than for granulocytes, as determined by flow cytometric analyses of rat and human leukocytes. Taken together, our findings in vivo indicate that the adhesive interactions responsible for rolling of PMNL and MNL, respectively, are distinct in terms of receptor occupancy, and may help explain the temporal selectivity in recruitment of different leukocyte subpopulations in inflammatory or immune reactions.

Effects of environmental stress on tissue survival and neutrophil recruitment in surgical skin flaps in relation to plasma corticosterone levels in the rat.

OBJECTIVE: Because glucocorticoid treatment can improve the survival of surgical skin flaps, we examined the influence of environmental stress on skin flap survival in the rat. MATERIAL: Female Sprague-Dawley rats. TREATMENT: Dexamethasone (1 mg/kg i.p.). METHODS: A standardized dorsal skin flap was raised and sutured back into position, and six days latter the percentage of flap survival was assessed. Corticosterone in rat plasma was measured using radioimmuno assay, and skin flap myeloperoxidase accumulation (reflecting neutrophil recruitment) was determined spectrophotometrically. RESULTS: Skin flap survival decreased gradually during a 10 day acclimatization period after transportation of the animals from the supplier, and plasma corticosterone levels were increased during the first 5 days of acclimatization compared to day 7 and 10. Dexamethasone treatment of rats accustomed to their new environment for 10 days increased flap survival to a level close to that observed in animals operated at day 1 after arrival. Flap surgery induced pronounced neutrophil recruitment into flap tissue, and this cell accumulation was greatly reduced in both the dexamethasone treated rats and in rats with elevated corticosterone levels. CONCLUSIONS: Skin flap survival in rats exposed to environmental stress may be significantly increased as compared to animals accustomed to their new environment for one week, possibly as a consequence of anti-inflammatory actions exerted by stress-induced elevations in plasma corticosterone. These findings emphasize the importance of strictly controlling environmental stress factors in studies of inflammation and tissue damage after surgical skin trauma.

Tumor cell arrest in the microcirculation: lack of evidence for a leukocyte-like rolling adhesive interaction with vascular endothelium in vivo.

Hematogenous spread of tumor cells and metastasis formation in secondary organs are insidious aspects of cancer. In the present intravital microscopic study in the rabbit mesentery, we examined the in vivo flow behavior of six human tumor cell lines of different histological origin. The tumor cells and human neutrophils were injected locally into a side branch of the superior mesenteric artery upstream of the observed microvascular area in the mesentery. None of the tumor cells behaved similar to the leukocytes of which a substantial fraction rolled along the endothelium of small venules. Thus, the tumor cells passed the same venular segments without interacting with the endothelial lining. Yet, three of the tumor cell lines (HT-29, DLD-1, and HCT-8) were strongly positive for the oligosaccharides Lewis(x), sialyl-Lewis(x), and sialyl-Lewis(a) which are recognized by the endothelial selectins that mediate leukocyte rolling. On the other hand, some tumor cells were trapped in the smallest vessels and remained so throughout the experimental period, apparently due to a discrepancy in size between tumor cells and microvessel lumen. Taken together, our in vivo findings suggest that initial microvascular arrest of metastasizing tumor cells is dependent primarily on mechanical factors rather than on receptor-mediated leukocyte-like adhesive interactions.

Impaired leucocyte rolling, adhesion and transendothelial migration following cuprophane haemodialysis.

In this study, we investigated how different steps in the extravasation process of leucocytes, i.e. rolling, adhesion and transendothelial migration, are affected by haemodialysis with cuprophane membranes. Human leucocytes obtained from whole blood prior to clinical haemodialysis and from the afferent blood line (post-dialyser) 15 min after the initiation of dialysis were injected into the mesenteric microcirculation of urethane anaesthetized rabbits and analysed for their ability to roll in the microvessels by use of intravital fluorescence microscopy. Moreover, neutrophils from the two leucocyte populations were compared with respect to chemoattractant-induced adhesion and transmigration across confluent monolayers of bovine aortic endothelial cells. Our results show that, as compared with pre-dialysis leucocytes, 15 min of cuprophane haemodialysis impaired leucocyte rolling by 78 +/- 7%, reduced N-formyl-methionyl-leucyl-phenylalanin (fMLP)-induced adhesion by 34 +/- 9%, and abolished transendothelial migration. These findings demonstrate that intradialytical activation of leucocytes during cuprophane haemodialysis severely affects leucocyte functions that are critical in the extravasation process of these cells at inflammatory tissue sites, and thus may help explain the increased susceptibility to infections observed in patients on chronic haemodialysis.

Microvascular actions of histamine: synergism with leukotriene B4 and role in allergic leucocyte recruitment.

BACKGROUND: Previous studies have shown that antihistamines provide little or no protection against the recruitment of leucocytes in allergic inflammation. OBJECTIVE: We wanted to examine if threshold doses of histamine can potentiate chemoattractant-induced leukocyte adhesion and if complete inhibition of histamine-induced microvascular effects is necessary to reduce allergic leucocyte recruitment. METHODS: The role of histamine in allergic leucocyte recruitment was examined by use of intravital microscopy of the hamster cheek pouch microcirculation. RESULTS: We found that topical administration of histamine caused a concentration-dependent increase in microvascular permeability in the cheek pouch; i.e. 0.3 microM histamine caused no detectable plasma leakage, while 1 microM and 10 microM histamine resulted in 29 +/- 9.3 and 356 +/- 47 leakage sites/cm2 cheek pouch area, respectively. The percentage of postcapillary venules with more than five adherent leucocytes (an index of early leucocyte recruitment) was 1.1 +/- 0.51% in the control situation, and did not increase significantly after stimulation with histamine alone (0.3-10 microM) or with 1 nM leukotriene B4 (LTB4). On the other hand, coapplication of 10 microM histamine and 1 nM LTB4 increased leucocyte adhesion 24-fold. In fact, the 10 times lower dose of histamine (1 microM) together with 1 nM LTB4 increased leucocyte adhesion to a similar extent (20 fold). The increase in vascular permeability evoked by exogenous 10 microM histamine (with or without LTB4), or by histamine released from activated mast cells (antigen challenge), was completely reversed by local pretreatment with the H1-receptor antagonist mepyramine. This mepyramine treatment also abolished the enhanced leucocyte adhesion in response to coapplication of histamine and LTB4. Moreover, mepyramine, which had no effect on leucocyte recruitment evoked by 3 nM LTB4 per se, reduced antigen-induced recruitment of leucocytes to the extravascular tissue by 79.5 +/- 14.8%. CONCLUSION: We conclude that threshold concentrations of histamine can strikingly potentiate chemoattractant-induced leucocyte responses, and that in order to reduce allergic leucocyte recruitment it may be necessary to use antihistamines in doses high enough to abolish the microvascular actions of histamine.

Cytokine-induced leukocyte rolling in mouse cremaster muscle arterioles in P-selectin dependent.

Leukocyte rolling and adhesion are generally observed in venules but rarely observed in arterioles. With the use of intravital microscopy, we found that a 4-h treatment with interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) dose dependently induced leukocyte rolling and adhesion in arterioles of the mouse cremaster muscle. The rolling response lasted more than 24 h and was completely inhibited by treatment with the sulfated polysaccharide fucoidin. Moreover, we found that costimulation with IL-1beta and TNF-alpha for 4 h synergistically increased arteriolar leukocyte rolling, i.e., threshold doses of IL-1beta and TNF-alpha together caused a more than 10-fold increase of rolling in arterioles compared with the sum of the individual responses. This rolling interaction was abolished by treatment with a monoclonal antibody directed against P-selectin (RB40.34), but it apparently was unaffected by a monoclonal antibody against L-selectin (MEL-14). Taken together, our functional data show that IL-1beta and TNF-alpha separately induce and synergistically increase P-selectin-dependent leukocyte rolling and firm adhesion in mouse cremaster arterioles.