Skin Window Research Articles

Leukotriene- and Histamine-Induced Increases in Vascular Permeability and Interstitial Transport in the Skin

The aim of the present study was to investigate the edema induced by leukotriene D4 (LTD4). Edema is the gross effect of increased blood flow and capillary permeability. Our objective was to elucidate the effect of LTD4 on edema generation without the influence of the concomitantly increased blood flow induced by this potent vasodilator. A new method was outlined measuring the exudation through skin windows of a macromolecular tracer [( 131I]albumin) and a micromolecular tracer [( 99Tc]pertechnetat). Exudation of [131I]albumin is a function of enhanced vascular permeability and interstitial transport and blood flow, whereas that of [99Tc] pertechnetat is mainly a function of blood flow. Thus a ratio of the 2 exudate measurements gives a specific estimate of the changes in vascular permeability and interstitial transport. Histamine was employed to establish the method. A dose-response relation was found for histamine in the dose range of 10(-4) to 10(-7) M. The effect of 10(-5) M LTD4 on vascular permeability and interstitial transport of macromolecules was indistinguishable from that of histamine in the same concentration. This property together with their well-known vasodilatory capabilities indicate that the leukotrienes of the slow-reacting substance of anaphylaxis may play a role in the vascular changes in the allergic-inflammatory reaction.

Granulocyte migration in ulcerative colitis.

Although histologically relapses in ulcerative colitis are characterized by an intense granulocyte infiltrate, there is evidence both from in vitro function tests and skin window migration studies in vivo that there is defective granulocyte function in ulcerative colitis. We have recently developed a method for non invasively monitoring granulocyte migration to sites of inflammation using indium-111-labelled granulocytes, and have used this technique in fifteen patients with ulcerative colitis. In all fifteen cases there was rapid migration of indium-111-labelled granulocytes to inflamed bowel within 10 min of return of the labelled cells. This study provides no support for a significant granulocyte migration defect in ulcerative colitis.

In vivo assessment of granulocyte migration to diseased bowel in Crohn's disease.

It has been suggested, on the basis of impaired granulocyte migration to skin windows, that there is a fundamental granulocyte defect in Crohn's disease. In vitro tests of granulocyte function have, however, failed to confirm this. We have studied granulocyte migration to inflamed bowel in Crohn's disease using a new approach which utilises dynamic gamma camera imaging after injection of 111In labelled autologous granulocytes. In 20 of 22 studies there was rapid migration to diseased bowel, compatible with no migration delay. Only two patients showed delays in migration of 12 and 15 minutes respectively, but neither had any clinical characteristics to distinguish them from the other 20 patients. This study shows that the majority of patients with Crohn's disease in relapse have rapid granulocyte migration to diseased bowel and provides evidence against a significant migration defect in this condition.

Investigations of host defence in patients with sickle cell disease.

Parameters of host defence were investigated in 30 patients with sickle cell disease (SCD). A newly devised perfusion system was used to study the kinetics in whole blood of leucocyte adherence, phagocytosis, killing and solubilization of a mixture of Staph. aureus and Str. pneumoniae, and secretion of lactoferrin. A skin window technique was used to examine the accumulation of leucocytes at inflammatory foci and their subsequent rate of movement through a filter. Serum concentrations of C3, C4, total haemolytic complement and immunoglobulins were also measured. The rate of neutrophil migration into filters was slightly reduced in patients with SCD. The proportion of monocytes that emigrated from the skin windows and their rate of migration were markedly diminished. The adhesion of neutrophils and their ability to kill staphylococci were also reduced, particularly in patients of the haemoglobin (Hb) SS and Hb S-beta-thalassaemia genotypes. Neutrophil function was mostly impaired in patients with the greatest frequency of bacterial infection. The rate of clearance of pneumococci was related to the concentration of type specific immunoglobulin G but not M. Serum concentrations of immunoglobulins and complement were normal. We were unable to define a defect of host defence of sufficient magnitude to explain the susceptibility of these patients to severe infection.

Ultrastructure of skin window cells in malignant lymphoma.

The ultrastructure of cells in skin window preparations was analysed in 38 patients with malignant lymphoma and in 22 normal subjects. In the series as a whole, there were significant morphological changes in the macrophages between day 1 and day 2 consistent with cell maturation during the inflammatory response. At both intervals, lymphoma patients showed strikingly larger cell and nuclear profile areas as compared with normal subjects; there was a greater number of nucleoli per profile and proportionately more sections showed a Golgi complex. At day 1, a greater percentage of the patients' macrophages showed large numbers of phagocytic inclusions, but at day 2, significantly fewer profiles demonstrated this feature. These morphological findings could be a reflection of the known mononuclear phagocyte dysfunction in malignant lymphoma.

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.

Defective neutrophil motility in patients with primary ciliary dyskinesia.

Microtubules are important in the regulation of the motile functions of a variety of cells, including leukocytes, ciliated cells and spermatozoa. Polymorphonuclear leukocyte function was studied in ten patients with primary ciliary dyskinesia, an inherited disorder of microtubules in sperm tails and cilia. Neutrophil chemotaxis in Boyden chambers was slightly reduced, but only one patient showed a migration below normal values. In vivo mobilization of polymorphonuclear leukocytes into skin windows was also slightly decreased. In contrast, neutrophil polarization and orientation was normal. The bactericidal activity of neutrophils from patients with primary ciliary dyskinesia was normal, while the ingestion of bacteria was decreased. The abnormalities of neutrophil function in patients with primary ciliary dyskinesia are related to motility. It is suggested that the microtubule defect responsible for the abnormal pattern of ciliary beating is a general abnormality also responsible for the depression of polymorphonuclear leukocyte motility.

Abnormal regulation of inflammatory skin responses in male patients with chronic granulomatous disease.

A common characteristic of the response to infection seen in patients with chronic granulomatous disease (CGD) is an exaggerated and prolonged inflammatory response with frequent development of draining lymph nodes and granuloma formation. Recent reports of several CGD patients with minor but significant in vitro abnormalities of cellular and humoral components of neutrophil chemotactic responses would predict lessened responses to inflammatory stimuli. The following studies were, therefore, performed to assess in vivo inflammatory responses in patients with CGD. Twenty-four-hour Rebuck skin-window procedures were performed on eight patients (five male and three female) with CGD and on ten volunteers. The windows were changed 1, 3, 5, 8, 12, and 24 h after the abrasion. Quantitation of the skin windows was performed with the assistance of a microscope-image analyzer computer facility. Neutrophil accumulation into skin windows was normal in CGD patients throughout the first 5 h. However, during the 8- to 24-h period, when neutrophils characteristically disappear from normal inflammatory responses and are replaced by monocytes, there was abnormal persistence of PMN at the inflammatory foci in male but not in female CGD patients (P less than 0.05 for the comparison of the rates of decline of PMN, from hour 8 to hour 24, in five male CGDs and in 10 normals). Monocyte recruitment was normal. In one CGD male, the abnormal skin-window response was normalized while he was receiving white cell transfusions. The data indicate that there is an abnormal "turn off" of the acute inflammatory response in male CGD patients and support a modulatory role for products of oxidative metabolism on the inflammatory response.(ABSTRACT TRUNCATED AT 250 WORDS)

In Vivo Mobilization of Polymorphonuclear Leukocytes in Psoriasis: Relationship to Clinical Parameters and Serum Inhibitory Factors

Mean migration of polymorphonuclear leukocytes (PMNL) toward autologous and homologous control sera, evaluated by quantitative skin window chamber technique, was only slightly reduced in 60 patients with psoriasis as compared to 27 normal controls (p less than 0.1). A significant decrease in cell migration was found (1) in patients with actively spreading lesions, (2) in patients with extensive lesions involving more than 40-60% of the skin surface, (3) in the first 2 months of relapse, and (4) 5-6 months after onset of new lesions. However, PMNL migration was increased when psoriatic lesions lasted 3-4 months. Seventy-one percent of psoriatic sera exerted a suppressive effect on the psoriatic and normal PMNL migration. The inhibitors were found predominantly in patients with stationary and long-standing lesions. Some of the psoriatic sera had a stimulatory effect on the chemotaxis of psoriatic PMNL. These sera originated from those patients with active spreading lesions in the first 2 months of relapse. These data indicate that neutrophil migration is abnormal in the course of psoriasis and that it could be modified by different proportions of both inhibitors and stimulators of chemotaxis.

The skin window as a monitor of leukocytic functions in contact activation factor deficiencies in man.

The skin window as a monitor of leukocytic functions in contact activation factor deficiencies in man.

Effects of storage of granulocytes on their fate in vivo.

During storage of granulocytes, the chemotactic response deteriorates first and to the greatest extent. Because in vitro assays such as chemataxis may not adequately predict the fate of granulocytes in vivo, we determined the intravascular kinetics and migration into skin windows of indium-111 labeled granulocytes. Granulocytes stored for 24 hours at either 1 to 6 or 20 to 24 degrees C had significantly reduced intravascular recoveries and migration into skin windows. Granulocytes stored for only eight hours at 20 to 24 degrees C had normal intravascular recovery and migration into skin windows. In granulocytes stored for eight hours at 1 to 6 degrees C, the intravascular recovery and migration into skin windows were reduced. Because of the method of calculating migration, this observation suggests that granulocytes which do circulate after storage at 1 to 6 degrees C had decreased ability to migrate into the skin windows. Granulocytes can be stored for up to eight hours at 20 to 24 degrees C without reduction in their ability to circulate and migrate to a site of inflammation. However, since many practical issues in granulocyte storage are not resolved, granulocytes should be transfused as soon as possible after collection.

Persistent mandibular infection in three patients with lazy and incompetent phagocyte syndromes

Three patients with the lazy phagocyte syndrome suffered from recurrent infections in the orofacial region which persisted despite treatment with antibiotics. All had neutrophil counts at the lower normal limits which did not increase after strenuous exercise or rise during infections. Tests of chemotaxis, random mobility and the skin window were abnormal. Ingestion and intracellular killing, however, were normal in one patient and abnormal in two. The findings are compatible with the lazy leucocyte syndrome but the symptoms were not manifest in early childhood as originally described by Miller et al. (1971), and the neutrophil counts were not as low as in his patients. Two of our patients also differed in showing a defective intracellular killing for Candida albicans. The lazy leucocyte syndrome should now be recognized to include different variants, some with late onset, a range in the degree of neutropenia, and some with defective ingestion and killing, though all possess a basic intrinsic defect of movement.

Severe recurrent bacterial infections associated with defective adherence and chemotaxis in two patients with neutrophils deficient in a cell-associated glycoprotein

We studied two patients with delayed umbilical cord detachment, recurrent bacterial infections, inability to form pus, rapidly progressive periodontitis, and persistent leukocytosis. The phagocytes of both patients were strikingly abnormal in their ability to adhere to surfaces. The adherence of polymorphonuclear leukocytes to endotoxin-coated glass coverslips, glass beads, or nylon wool was markedly reduced. Scanning electron microscopy of the few adherent polymorphonuclear leukocytes from both patients showed a failure to flatten and form fine pseudopods. In vivo polymorphonuclear leukocyte and monocyte chemotaxis assessed by skin window and skin chamber methods was dramatically impaired, and in vitro chemotaxis was severely depressed. Chemiluminescence of zymosan- but not phorbol-stimulated polymorphonuclear leukocytes was markedly reduced. Allogeneic polymorphonuclear leukocytes transfused into these patients functional normally, indicating that the defect is intrinsic to the cells and not a secondary phenomenon. A 180-kilodalton glycoprotein normally present in the particulate fraction of polymorphonuclear leukocytes was found to be completely absent in Patient 1 and present in low concentration in Patient 2. We postulate that the glycoprotein deficiency interferes with the migration of polymorphonuclear leukocytes from the bloodstream into the interstitial space and to the site of infection.

FUNCTION OF POLYMORPHONUCLEAR NEUTROPHILIC LEUCOCYTES

The function of polymorphonuclear neutrophils from blood (B-PMN) and from exudates (E-PMN) was studied in healthy volunteers. The E-PMNs were isolated from skin windows with chambers and the chemotactic, phagocytic and Nitro Blue Tetrazolium (NBT) reducing activity measured and compared to that of simultaneously obtained B-PMNs. The chemotactic and random migration of E-PMNs in Boyden chambers measured by the leading front and by the chemotactic index were reduced compared with B-PMNs (p less than 0.01). Serum independent phagocytosis of paraffin oil emulsions was significantly increased (p less than 0.01) by E-PMNs after 12, 24 and 48 hours and not correlated to the function of B-PMNs. Opsonization with autologous serum increased the phagocytosis by both E-PMNs and B-PMNs, but mainly the latter. The NBT reduction by E-PMNs was increased (p less than 0.01) and positively correlated to the NBT reduction by resting B-PMNs. In contrast, there was no difference in NBT reduction between phagocytosing E-PMNs and B-PMNs. Mobilization of PMNs to an inflammatory focus in healthy subjects induces marked changes in the function, and B-PMN function cannot always be assumed to reflect the function of PMNs from inflammatory sites.

Leucocyte function in ulcerative colitis. Quantitative leucocyte mobilisation to skin windows and in vitro function of blood leucocytes.

Leucocyte function was evaluated by the in vivo mobilisation to skin windows with chambers and by the chemotactic, phagocytic, and nitro blue tetrazolium reducing capacity of blood leucocytes from 20 patients with ulcerative colitis. The total number of leucocytes mobilised to the chambers after 12 hours did not differ from those in 21 healthy volunteers. After 24 and 48 hours reduced number of leucocytes were mobilised by patients with ulcerative colitis (p less than 0.01). Correspondingly, the leucocyte migration rates were normal initially but were reduced after 18 hours Mobilisation in vivo was positively correlated to the blood neutrophil count (Rho:0.5549 po less than 0.02) but unrelated to clinical activity. Blood leucocytes showed reduced random migration in vitro as well as chemotactic response to casein (p less than 0.01). Serum independent and dependent phagocytosis did not differ from healthy volunteers. Nitro blue tetrazolium reduction by resting leucocytes was increased (p less than 0.01) in ulcerative colitis compared with controls. Our findings suggest altered in vivo mobilisation and in vitro migration of leucocytes in ulcerative colitis with increased spontaneous nitro blue tetrazolium reduction reflecting increased generation of potentially tissue damaging agents. The findings probably reflect changes secondary to the disease but which may be important in maintaining the inflammatory process.

Generalized monocyte deficiency in leukaemic reticuloendotheliosis.

Examination of the blood of 40 patients with leukaemic reticuloendotheliosis (LRE) revealed a drastic reduction in monocyte numbers (mean 74/microliter) when compared to a normal group of 33 (mean 442/microliter). Repeated blood studies in 8 LRE patients were made over periods of 2 months to 5 years and monocytopenia was observed to be a persistent phenomenon. Similarly, tissues from 29 patients showed an overall decrease in numbers of monocytes/ histiocytes when compared with those of 73 control subjects. Skin window examinations in all 11 patients so studied showed marked reduction or absence of monocyte response to inflammation. 2 exceptional cases to this trend are noted. In one, circulating monocyte levels remained normal over a 14-month period while examinations of blood and marrow in this case showed few hairy cells. In another case, severe and persistent monocyte deficiency was noted during a 2-year period of unsuccessful treatment. When he eventually responded to low dose chlorambucil and his Hb rose from 40-140 g/l and WBC became normal, the number of circulating monocytes also became normal.

Human Neutrophil-Specific Granule Deficiency: A Model to Assess the Role of Neutrophil-Specific Granules in the Evolution of the Inflammatory Response

It has been suggested that neutrophil (PMN) specific granules are important in cell aggregation, locomotion, hydroxyl radical formation, and in extracellular functions such as the generation of complement-related inflammatory mediators (C5a) and the feedback regulation of myelopoiesis. In the current studies, a 9-yr-old boy with a history of recurrent infections and specific granule deficiency (absent lactoferrin, B-12 binding proteins, and characteristic specific granules on sucrose gradient centrifugation of cell homogenates) was studied to assess some of these concepts. In vivo, the patient had decreased PMN and monocyte accumulation into Rebuck skin windows but an expected febrile episode with an associated neutropenia (PMN margination) and neutrophilia (mobilization of marrow reserves) in response to intravenous endotoxin. In vitro, the patient's resting PMN showed increased ruffling, increased surface-to-volume ratio, and increased numbers of centriole-associated microtubules. His PMN showed a significant decrease in cell negative surface charge (which may relate to aggregation) in response to several stimuli and adhered better than normally to plastic. In addition, his PMN aggregated normally in response to the chemoattractant f-met-leu-phe, although the subsequent disaggregation normally seen with PMN did not occur with the patient's cells. Chemotaxis of the patient's PMN to several stimuli was abnormal, and specific saturable and displaceable binding of the chemoattractant f-met-leu-[3H]phe was decreased. Similarly, following incubation with secretagogues, there was a less than normal increase in f-met-leu-[3H]phe binding and an absence of the normal increases in PMN surface area. The patient's PMN bactericidal activity, stimulated oxygen metabolism (cytochrome-c reduction, chemiluminescence, and NBT reduction), and elicited changes in membrane potential were also abnormal. Studies assessing the mechanism for the abnormal monocyte accumulation into skin windows indicated the patient's monocyte chemotaxis was better than normal in vitro. However, the patient's PMN homogenates lacked a stimulus of monocyte locomotion and did not generate chemotactic activity normally from serum. Thus, the data indicate that specific granule constituents are not required for neutrophil margination in vivo or aggregation in vitro. However, the data support the concept that PMN-specific granules are important for PMN locomotion and oxidative metabolism. In addition, extracellular release of specific granule constituents appears to be important for amplification of the initial and subsequent phases of the inflammatory response.

Human neutrophil-specific granule deficiency: a model to assess the role of neutrophil-specific granules in the evolution of the inflammatory response

It has been suggested that neutrophil (PMN) specific granules are important in cell aggregation, locomotion, hydroxyl radical formation, and in extracellular functions such as the generation of complement- related inflammatory mediators (C5a) and the feedback regulation of myelopoiesis. In the current studies, a 9-yr-old boy with a history of recurrent infections and specific granule deficiency (absent lactoferrin, B-12 binding proteins, and characteristic specific granules on sucrose gradient centrifugation of cell homogenates) was studied to assess some of these concepts. In vivo, the patient had decreased PMN and monocyte accumulation into Rebuck skin windows but an expected febrile episode with an associated neutropenia (PMN margination) and neutrophilia (mobilization of marrow reserves) in response to intravenous endotoxin. In vitro, the patient's resting PMN showed increased ruffling, increased surface-to-volume ratio, and increased numbers of centriole-associated microtubules. His PMN showed a significant decrease in cell negative surface charge (which may relate to aggregation) in response to several stimuli and adhered better than normally to plastic. In addition, his PMN aggregated normally in response to the chemoattractant f-met-leu-phe, although the subsequent disaggregation normally seen with PMN did not occur with the patients cells. Chemotaxis of the patient's PMN to several stimuli was abnormal, and specific saturable and displaceable binding of the chemoattractant f-met-leu-[3H]phe was decreased. Similarly, following incubation with secretagogues, there was a less than normal increase in f-met-leu-[3H]phe binding and an absence of the normal increases in PMN surface area. The patient's PMN bactericidal activity, stimulated oxygen metabolism (cytochrome-c reduction, chemiluminescence, and NBT reduction), and elicited changes in membrane potential were also abnormal. Studies assessing the mechanism for the abnormal monocyte accumulation into skin windows indicated the patient's monocyte chemotaxis was better than normal in vitro. However, the patient's PMN homogenates lacked a stimulus of monocyte locomotion and did not generate chemotactic activity normally from serum. Thus, the data indicate that specific granule constituents are not required for neutrophil margination in vivo or aggregation in vitro. However, the data support the concept that PMN-specific granules are important for PMN locomotion and oxidative metabolism. In addition, extracellular release of specific granule constituents appears to be important for amplification of the initial and subsequent phases of the inflammatory response.

Serum inhibitors of leukocyte chemotaxis in Crohn's disease and ulcerative colitis

Studies were undertaken to determine the incidence and nature of serum chemotaxis inhibitors in patients with Crohn's disease and ulcerative colitis. Sera from patients with active Crohn's disease were shown to contain chemotactic factor-directed inhibitory activity. The molecular size of the inhibitor estimated by gel filtration is similar to the 7S γ-globulins and it is precipitable by 40% ammonium sulfate. Less marked inhibitory activity is present in the same fractions of sera from patients with ulcerative colitis and from healthy controls, suggesting that it represents increased activity of one or more normal serum globulins. Leukocyte-directed serum inhibitors have also been demonstrated in patients with Crohn's disease and ulcerative colitis, particularly in patients with active disease. In Crohn's disease the presence of serum chemotactic factor inhibitors may explain the diminished chemotaxis of neutrophils into skin window chambers reported previously. The inhibitors are nonspecific and are probably secondary to the disease state but may have some bearing on the nature of the mucosal inflammation.

The chemotactic function of neutrophils in ulcerative colitis.

Neutrophil chemotactic function was investigated in 30 patients with ulcerative colitis (UC) in an active (no. = 15) or an inactive (no. = 15) stage. Peripheral blood neutrophils from active cases had a significantly decreased stimulated migration in modified Boyden chambers. Simultaneously done investigations of neutrophil migration into skin window chambers showed normal results in both active and inactive cases of UC. Thus, the defective chemotaxis during in vitro conditions was not reflected in a quantifiable neutrophil dysfunction in experimental acute inflammation.