Interstitial Mast Cells Research Articles

Histamine-containing lung cell structures in certain forms of secondary tuberculosis

The aim of research was to study the distribution of histamine in macrophages and mast cells of the lung structures in autopsy material in certain forms of secondary tuberculosis.Material and methods. The authors examined 165 cases of secondary pulmonary tuberculosis. The study included 500 histological specimens of male patients, aged 30 to 55 years, divided into 5 groups depending on the forms of secondary tuberculosis., The luminescent-histochemical method of Cross was used to determine the content of histamine in the lung structures; the specimens were further exposed to re-staining with hematoxylin and eosin.Results. Histamine-containing specific alveolar and interstitial macrophages and mast cells were detected when studying autopsy material from the lungs of "nominally healthy" people. The highest content of histamine in the lung structures of this group was recorded in interstitial macrophages. In acute focal tuberculosis, there was an increased content of histamine in intra-alveolar and interstitial macrophages, with a simultaneous decrease of this bioamine in mast cells. In fibro-focal tuberculosis, the content of histamine exceeded the control parameters in all the studied cellular lung structures, with the highest content in mast cells. In infiltrative tuberculosis, there was detected an infiltrative luminescent track from lymphocytes together with the maximum luminescence of histamine. In acute cavernous and fibrous-cavernous tuberculosis, total degranulation of mast cells was observed. The content of the studied bioamine significantly decreased in interstitial macrophages compared to the control group.Conclusion. The study of small forms of secondary tuberculosis detected an increased concentration of histamine in intra-alveolar and interstitial macrophages. Its maximum accumulation in all cellular structures was registered in the infiltrative form of tuberculosis. In destructive forms of tuberculosis, histamine was detected only in macrophages, which appeared to be the regulatory link of specific inflammation. The literature data and the results obtained allow us to report on the influence of Mycobacterium tuberculosis as a corpuscular antigen that causes destruction of structures that produce and metabolize neuroamines in the lungs.

Morphological analysis of interstitial Cajal cells and mast cells in experimental hyperactivity bladder and stress incontinence under influence of pharmacocorrection

The existing data indicate the multifactorial mechanisms of development of the overactive bladder (OAB) symptom, but the issue of OAB pathogenesis remains unclear. In more recent times, the neurogenic theory of OAB genesis has being accompanied by the increasing attention to the study of morphological changes that occur in the smooth myocytes of the detrusor and their interaction with the extracellular matrix. Therefore, the objective of our study became the evaluation of distribution of interstitial Cajal cells (ICC) and basophilic granulocytes (BG) in the structural elements of the bladder wall under stress urinary incontinence and its overactivity before and after treatment with Mirabegron, Spasmex, Quercetin and combination thereof with testosterone and estradiol, using histochemical and immunohistochemical methods. The experimental models of OAB and stress urinary incontinence (SUI) presented the increase in the amount and functional activity of BG revealed by histological and immunohistochemical methods, as well as ICC at all terms of OAB monitoring, while the SUI presented with high concentration and functional activity of BG only after 14 days of the experiment. After 28 days, we observed a sharp decrease of the parameters, indicating decompensation and depletion of the functional activity. The number of ICC decreased under SUI after both 14 days and 28 days of the experiment. The group of experimental animals receiving Spasmex and its combination with hormones, presented no significant effect on the quantitative and qualitative composition of BG and ICC at OAB and SUI on Day 14 of the experiment, but the combination with testosterone demonstrated statistically reliable (p<0.001) reduction of BG and ICC expression in the muscle layer of the bladder after 14 days, and unreliable after 28 days of the experiment (p>0.05). The administration of Mirabegron alone and especially its combination with testosterone and estradiol presented positive trends in histochemical and immunohistochemical expression of BG and ICC. The experiment proved high efficacy of Quercetin in combination with testosterone and estradiol under OAB and SUI, confirmed by stabilization of the functional activity of BG and ICC quantitative composition.

PATHOMORPHOLOGY OF SKELETAL MUSCULATOR AND MICROCIRCULATORY VASCULAR BED IN EXPERIMENTAL HYPOTHERMISM

The article describes the results of studying the effect of hypothermia on skeletal muscles and microcirculatory vascular bed of rats in the experiment. Hypothermia was modeled by placing animals in individual cells in water at the temperature of 5 °C, at an ambient temperature of 7 °C. It is shown that along with the damaging effect on muscle tissue and microcirculatory vascular bed, hypothermia is a powerful activator of the mast cells’ and macrophages’ activity. Interstitial mast cells and macrophages are important factors in the process of adaptation of the body to the effect of hypothermia.

TNF-α AND IL-10 GENE POLYMORPHISMS VERSUS CARDIOIMMUNOLOGICAL RESPONSES IN SUDDEN INFANT DEATH

We hypothesized that genetic variations of cytokines could contribute to the risk of developing a fatal immunological reaction in the heart of infants. Thus, tumor necrosis factor (TNF)-α and interleukin (IL)-10 gene polymorphisms versus induction of cardioimmunologxical responses in victims of sudden infant death syndrome (SIDS) were explored. We genotyped 35 infants (23 cases of SIDS and 12 infants with a known cause of death), and 100 healthy adult controls for IL-10 –1082 G/A, −592 C/A and TNF-α-238 G/A, −308 G/A. We found a higher frequency of the ATA haplotype and ATA/ATA genotype of IL-10 associated with SIDS (13%). The frequency of homozygote infants for IL-10 haplotypes in SIDS was higher (52%) than the control group (34%). All SIDS cases were homozygotice for the TNF-α-238 G allele and 20 infants were homozygous for the TNF-α-308 G allele in the same group. None of the infants with higher levels of infiltrated T-cells (n=8) was homozygous for IL-10 gene polymorphisms, whereas in contrast 3 cases of the 6 that displayed higher levels of cardiac mast cells were homozygous. In this study, the increased number of interstitial T-cells, mast cells, and macrophages in the myocardial interstitium demonstrated no correlation with the genotype for either cytokines.

Mast cells in testicular biopsies of azoospermic men

This work aimed at identifying mast cells in testicular biopsies from 10 normal fertile controls, 20 patients with obstructive azoospermia and 70 patients with nonobstructive azoospermia. The biopsies were stained with haematoxylin and eosin stain for tubular-modified Johnson score and with toluidine blue stain for mast cells. Two populations of mast cells, peritubular and interstitial, were demonstrated in all sections with varied counts. Testicular sections with Sertoli cell only and spermatogenic arrest patterns demonstrated a significant increase in both peritubular and interstitial mast cells compared with other groups, whereas obstructive azoospermia demonstrated a nonsignificant increase compared with the controls. Mast cell count was significantly correlated negatively with Johnson score for both peritubular (P = 0.001) and interstitial (P = 0.001) populations. Whether these results could be a cause or an effect, a special role might be assigned to mast cells in the pathogenesis of disturbed spermatogenesis.

Smooth muscle hamartoma of the hard palate

A small polypoid lesion was removed from the incisive papilla region of a healthy female aged 8 years. It was composed of non-cycling smooth muscle fibres that were immunoreactive for various muscle antigens, surrounded by basement membrane containing collagen IV, arranged as bundles and mixed with CD34(+) interstitial cells, mast cells, dendrocytes and nerves. The lesion could be attributed to dysgenesis affecting media of vessels emerging from the incisive foramen. Intra-oral smooth muscle hamartomas tend to favour regions featuring complex developmental events.

Computational Modeling of Enzymatic Nitric Oxide Production And Transport In The Microvasculature

Nitric oxide (NO) can be released from multiple enzymatic and non-enzymatic sources to regulate O2 delivery. In this study, we formulated computational kinetic models to predict NO production rates from specific NO synthase (NOS) sources. Based on the analysis of biochemical pathways of neuronal NOS (NOS1) and endothelial NOS (NOS3) catalytic activities and the availability of NOS isoforms and substrates, our model predicted that the NO production rate from vascular endothelium expressing NOS3 is 0.005 – 0.1 μM/s and the rate from the nearby nerve tissues and interstitial mast cells expressing NOS1 is 0.4 – 1.2 μM/s under 100 μM O2 condition. We also calculated [NO] distribution around an arteriole with the predicted NO production rates and the topographical information on NOS isoforms. From our model, the major source determining the distribution of NO was NOS1 in the nerve fibers and mast cells close to arterioles, rather than the endothelial NOS3. Our models also predicted that O2 reduction significantly limits NO formation from NOS1 and thus NO exposure of vascular smooth muscle, opening the possibility that non-enzymatic NO becomes an important source under hypoxia. Moreover, the perivascular [NO] predicted by the models under normoxia was an order of magnitude lower than a number of experimental measurements, suggesting the existence of other enzymatic or non-enzymatic sources of NO in the microvasculature.

Role of mast cells in the development of renal fibrosis: Use of mast cell–deficient rats

Recent clinical studies have shown that the number of interstitial mast cells increases in various types of renal disease and correlates well with the magnitude of interstitial fibrosis. The present study was conducted to assess the role of mast cells in renal fibrosis by examining an experimental glomerular disease. A rat model of chronic glomerular disease, puromycin aminonucleoside-nephrosis, was induced in mast cell-deficient (Ws/Ws) and normal (+/+) rats. The area of interstitial fibrosis was widely distributed at 6 weeks in both groups of rats; however, unexpectedly, the area of interstitial fibrosis was greater in Ws/Ws rats than in +/+ littermates. Biochemical analysis of the hydroxyproline content confirmed the more severe fibrosis in the Ws/Ws rats. The number of mast cells increased in both Ws/Ws and +/+ rats, concomitant with the development of interstitial fibrosis, but was confirmed to be lower in Ws/Ws than in +/+ rats. There were no differences in the numbers of interstitial macrophages and T lymphocytes between the two groups. Reverse transcription-polymerase chain reaction analysis of cytokine expression revealed that the level of mRNA for transforming growth factor-beta (TGF-beta), a potent profibrotic cytokine, was higher in Ws/Ws rats. In addition, heparin, one of the major components of mast cells, inhibited the expression of TGF-beta mRNA in rat fibroblasts in culture. These results suggest that mast cells do not play a major role in the pathogenesis of interstitial fibrosis in puromycin aminonucleoside nephrosis. Rather, they might be protective or ameliorative in this model through the inhibition of TGF-beta production by heparin, and possibly in other models and also in humans.

Stem cell factor and crescentic glomerulonephritis

Background: Numerous cells and cytokines have been implicated in the pathogenesis of crescentic glomerulonephritis (CGN). Recently, there has been growing awareness of the role of mast cells and their growth factor, stem cell factor (SCF), in the process of tissue inflammation and fibrosis. Methods: In this study, renal biopsy specimens from 28 patients with a histopathologic diagnosis of CGN were evaluated immunohistochemically for the presence of mast cells, SCF, and its receptor (c-kit). In addition, CD34+ hematopoietic cells, monocytes (CD68+ cells), and myofibroblasts (α-smooth muscle actin-positive [α-SMA+] cells) were counted. Renal biopsy specimens from cadaveric kidney donors and kidneys removed for hypernephroma (n = 6) served as controls. Point counting of positive immunostaining for SCF, α-SMA, and collagens III and IV was undertaken. Glomerular and interstitial fibrosis (IF) scores were determined. Results: Patients who developed progressive chronic kidney failure showed a significant increase in percentage of crescents, number of interstitial c-kit+ cells, and glomerular CD68+ cells compared with those with a favorable outcome. Analysis showed a significant elevation of tryptase+ mast cells in the interstitium of renal biopsy specimens of patients with CGN compared with controls. SCF and c-kit+ cells were found in glomeruli and interstitium, with occasional immunostaining of the crescent with SCF. Both glomerular and interstitial SCF immunostaining was significantly higher in biopsy specimens of patients compared with controls. Glomerular and interstitial SCF showed a significant positive correlation with 24-hour urinary protein level. There were a few CD34+ cells in both glomeruli and interstitium, but their numbers did not differ between patients and controls. Colocalization of CD34+ and c-kit+ was seen in some rounded interstitial and spindle-shaped cells. Number of interstitial mast cells proved to be a strong predictor of IF. Glomerular SCF correlated negatively with creatinine clearance and positively with glomerular CD68+ cells. Interstitial immunostainable SCF correlated positively with interstitial CD68+ cells and interstitial collagen III. On double antigen labeling, SCF was shown in the vicinity of α-SMA+ cells. Conclusion: These results show the potential involvement of mast cells and their growth factor SCF/c-kit in CGN.

Mast cells and fibrosis on testicular biopsies in male infertility.

Testicular dysfunction correlates with increased testicular mast cells. Mast cells can activate fibroblasts and promote collagen synthesis. The aim of the study was to examine testicular mast cells containing tryptase, and the relationship between mast cells and different fibrosis stages of interstitium and peritubular region of testes. Testicular biopsies obtained from 33 infertile men were assigned to 2 groups: normal spermatogenesis (n = 10) and defective spermatogenesis (n = 23). Total, interstitial, and peritubular mast cells were examined immunohistochemically using antihuman tryptase. The fibrosis stage was evaluated using vimentin and alpha-smooth muscle actin. The ratio of tubules with sclerosis to total tubules was also calculated. In all cases, mast cells were mainly localized in the interstitium. The number of total mast cells was significantly higher in defective spermatogenesis than in normal spermatogenesis (p = .048). In both groups, interstitial mast cells were higher than peritubular mast cells. However, the increase in peritubular region was much higher than the increase in interstitium. Total, peritubular, and interstitial mast cell counts were not different from each other, according to the changing fibrosis stages. Total and interstitial mast cells were significantly higher in the cases with sclerosing seminiferous tubules than in the cases with no sclerosis (p = .04 and p = .024, respectively). The mast cells and the mast cell product tryptase could be involved in the etiology of defective spermatogenesis, especially whenever the last stage (tubular hyalinization and sclerosis) takes place.

Quantitative analysis of interstitial mast cells in AA and AL renal amyloidosis.

Eighteen renal biopsy specimens obtained from patients with AA-type renal amyloidosis (AA) and 11 from patients with AL-type renal amyloidosis (AL), for whom both light and electron microscopy as well as immunofluorescence microscopy and full clinical data were available, were examined quantitatively. The cases were selected on the basis of immunohistochemical studies. As a control, we used 10 biopsy specimens from the kidneys removed because of trauma. Morphometric investigations were carried out by a computer image analysis system to find an answer to the question of whether mast cells can correlate with tubulointerstitial fibrosis in AA and AL renal amyloidosis, and to examine the relationship between mast cells and interstitial alpha-smooth muscle actin (alpha-SMA) expression and interstitial infiltrates. The morphometric study revealed that the mean values of the interstitial tryptase-positive cells, expression of alpha-SMA, interstitial volume, CD68+, CD45RB+, CD43+ and CD20+ cells were increased in AA as compared with the AL group, most of them significantly. Most of these parameters were also significantly increased in both AA and AL patients as compared with the control group. In both the AA group and the AL group, there existed some significant positive correlations between interstitial tryptase-positive cells and interstitial expression of alpha-SMA, interstitial volume and CD68+ cells. Interestingly, in AA cases, but not in AL cases, we noted a significant relationship between interstitial tryptase-positive cells and CD43+ cells. Our findings demonstrate that mast cells belong to the constitutive cell types in the interstitium in renal amyloidosis, in particular in amyloid type A. In addition, in both the AA group and the AL group, the significant positive correlations between interstitial mast cell count and relative interstitial volume and interstitial expression of alpha-SMA suggest that these cells play a role in the development of interstitial fibrosis.

Mast cells in acute and chronic rejection of rat cardiac allografts--a major source of basic fibroblast growth factor.

Studies of cardiac allograft arteriosclerosis, i.e., chronic rejection, have largely focused on mononuclear inflammatory cell infiltrates in the vascular wall and periphery of the occluded vessels. The purpose of this study was to investigate the role of mast cells in the development of acute and chronic rejection in rat cardiac allografts. In the acute rejection model, transplant recipients were not treated with immunosuppressants, and the grafts were removed 5 days after transplantation at the time of severe acute rejection. In the chronic rejection model, the recipients were administered triple-drug immunosuppression, and the grafts were removed 90 days after transplantation. During acute rejection, the number of mast cells was not increased, but the localization pattern differed from that of syngeneic grafts. In acute rejection, mast cells were located in the perivascular region of the allografts, but in syngeneic grafts, mast cells had a more interstitial location. In the chronic rejection model, the cardiac allografts with severe intimal thickening showed large numbers of mast cells at perivascular sites of occluded intramyocardial vessels and in the interstitium. Linear regression analysis revealed a significant correlation between the numbers of perivascular and interstitial mast cells and the intensity of intimal thickening. The majority of mast cells showed positive immunoreactivity to basic fibroblast growth factor (bFGF). Macrophage bFGF expression was not so prominent, but macrophages were more frequent in numbers. Tumor necrosis factor-alpha expression was detected mainly in macrophages and in only a few mast cells. When the intensity of arteriosclerosis was decreased by an increase in the intensity of immunosuppression, the numbers of intragraft mast cells and other mononuclear cells, and also the production of their respective cytokines, bFGF and tumor necrosis factor-alpha, gradually diminished. Taken together, our data show that the intensity of intramyocardial mast cell infiltration was associated with the intensity of chronic inflammation and allograft arteriosclerotic changes, but not with acute rejection, and that mast cells, in addition to macrophages, are a major source of myocardial bFGF. The results also demonstrate that when the T-cell activation pathway is blocked using cyclosporin, the number of mast cells is decreased. Cyclosporin may have affected the cytokine production that interfered with both the mast cell-dependent initiation and the leukocyte- and mast cell-dependent amplification and progression of the immune responses influenced by mast cell-leukocyte cytokine cascades. bFGF produced by mast cells may contribute to enhanced inflammation, neovascularization, and fibrosis during cardiac allograft arteriosclerosis.

IL-4 promotor gene polymorphism in heart transplantation

IL-4 is a cytokine of the Th2 subtype, secreted mainly by activated T- lymphocytes. IL-4 acts by down regulation of Th1 responses of macrophage and Th1 cells. On the other hand, IL-4 stimulates growth and differentiation of Th2 lymphocytes and mast cells. In transplantation Th2 cells are believed to be involved in induction of graft tolerance. Previous studies revealed that patients with a high frequency of IL-4 producing precursor T- helper cells (pHTL) before heart transplantation (HTX) had no or less rejection episodes compared to patients with low frequency of IL-4 producing pHTL (van Hoffen et al., Transpl Int. 13,S216,2000). Recently, single nucleotide polymorphisms (SNP) in the promoter regions of various cytokine genes have been identified. These SNP influence promoter strength and hence production of the cytokine. The promoter region of the IL-4 gene contains three SNP at positions -590 (C>T), -285 (C>T) and -81 (A>G) from the transcriptional start site. Three polymorphic alleles (resp T, T and G) increase promoter strength. We hypothesize that presence of these polymorphic alleles decreases rejection by increasing IL-4 production. Seventy HTX patients and 61 donors were genotyped for all three SNP by sequencing. SNP at -285 and -81 were not polymorphic in this study. We found 66 % CC, 30% CT and 4% TT at position -590 in patients and 80% CC, 18% CT, 2%TT in donors. Our results show that the incidence of rejection is significantly lower in patients receiving the T-allele from their donor compared to patients who did not ( x2 = 4.52, p= 0.034, relative risk = 3.31), regardless of the patients own haplotype. This indicates that IL-4 production within the donor heart and by cells from that donor is important. Potential candidates for this IL-4 production are various stromal elements, but also interstitial mast cells may be responsible.

Increased Density of Interstitial Mast Cells in Amyloid A Renal Amyloidosis

Renal interstitial fibrosis is the final common pathway leading to end-stage renal disease in various nephropathies including renal amyloidosis. However, the role of mast cells (MCs) in the fibrotic process of renal amyloidosis is not fully understood. We compared the distribution of MCs in renal biopsies from 30 patients with AA type renal amyloidosis and 20 control cases. Immunoreactivity of renal MCs to anti-tryptase and anti-chymase was studied. Interstitial myofibroblasts were stained with anti-α-smooth muscle actin (α-SMA) antibody, and inflammatory cells were identified by anti-CD45, -CD20, and -CD68 mAbs. Positively stained cells were counted, and the relative interstitial and fractional areas of anti-α-SMA stained cells were measured. Anti-CD29 mAb was used to detect β1 integrin and anti-basic fibroblast growth factor (bFGF) mAb for the growth factor on MCs. MCs were rarely found in control samples. In contrast, samples showing amyloid deposition contained numerous tryptase-positive (MCT) (940.17 ± 5.4 versus 6.74 ± 1.1/mm2) but fewer chymase-positive (MCTC) cells (20.7 ± 2.86 versus 1.7 ± 0.76/mm2) in the renal interstitium. There was a significant relationship between interstitial MCT and creatinine clearance (r = −0.72), and between interstitial MCT and glomerular amyloid-index (GAI) (r = 0.723) and interstitial amyloid area (r = 0.824). Accumulation of MCs correlated significantly with the number of T lymphocytes (MCT: r = 0.694). There was also a significant relationship between mast cell (MC) number and the fractional area of α-SMA positive interstitium (r = 0.733) and interstitial fibrotic area (r = 0.6). Double immunostaining demonstrated intracytoplasmic presence of β1 integrin on 87% of MCT and correlated significantly with the interstitial amyloid area (r = 0.818, P = .001) and T-cell number (r = 0.639, P = .002). bFGF was also detected on 85.5% of MCTC correlating well with the interstitial α-SMA-area (r = 0.789). Our results indicate that MCs constitute an integral part of the overall inflammatory process and play a crucial role in interstitial fibrosis in renal amyloidosis.

Mast cells and type VIII collagen in human diabetic nephropathy.

Renal injury in diabetes mellitus is associated with progressive interstitial fibrosis and extracellular matrix accumulation. However, the phenotypes of cells forming the interstitial infiltrate in diabetic nephropathy have not been precisely defined. There is increasing evidence for the association of mast cells with angiogenesis, chronic inflammatory conditions and fibrosis. We have recently shown that human mast cells can produce the non-fibrillar short chain type VIII collagen in vivo. Using immunohistochemistry, in situ hybridisation and reverse transcriptase-polymerase chain reaction, we examined the contribution of mast cells and type VIII collagen to the fibrotic changes occurring in biopsy-proven diabetic nephropathy. We observed that the number of interstitial mast cells was significantly increased in diabetic nephropathy compared with normal kidney tissue. In specimens from diabetic subjects, intense immunohistochemical staining for type VIII collagen was detected in mast cells, on periglomerular fibres and in perivascular and interstitial sites. The expression of type VIII collagen in periglomerular and interstitial sites coincided with that of alpha smooth muscle actin, a marker for myofibroblastic differentiation mRNA for type VIII collagen was detected by reverse transcriptase-polymerase chain reaction in diabetic nephropathy and in a human mast cell line. By in situ hybridisation the transcripts for type VIII collagen were localised to renal mast cells. The increased number of mast cells and the elevated type VIII collagen deposition in human diabetic nephropathy provides a potential link between the extracellular matrix accumulation and the fibrosis observed in this condition.

Transvascular and interstitial migration of neutrophils in rat mesentery.

The consequential implications of the leukocyte-endothelial cell adhesion reaction, as well as the subsequent migratory activity of the leukocytes within the interstitium proper, are currently known only in general phenomenological terms. Our objective was to carry out a detailed quantitative analysis of transendothelial and interstitial migration. We incorporated several new features, such as the video recording of time-lapse photography in conjunction with the time history of individual leukocytes under the influence of representative stimulators and inhibitors. The mesentery was suffused with the chemical activator N-formyl-met-leu-phe (fMLP, 5 x 10(-8) M) and histamine (10(-6) M) for periods up to 3 h. This leads to an attachment of neutrophils preferentially to the walls of small postcapillary venules followed by migration into the adjacent interstitium. The time interval between the initial neutrophil adhesion to the luminal side of the venules and their first appearance on the abluminal side of the venule wall was about 25 min. The time interval for the actual physical migration across the endothelium was only about 1-2 min. There was an initial increase in the number of neutrophils migrating across the wall per unit time after the application of fMLP. However, this effect then gradually decreased over the course of 1 h. In the extravascular tissue, the neutrophils appeared to migrate along random pathways with an average trajectory away from the venular wall. When histamine (1 microM) was applied topically in conjunction with fMLP, the overall migratory flux across the venular wall increased, although the cells migrated comparatively short distances into the interstitium. After pretreatment with phalloidin (25 micrograms/kg), an actin cross-linking agent, neutrophil adhesion and migration in response to fMLP stimulation was reduced. The mast cell stimulator 48/80 (25 mg/ml) resulted in rapid degranulation, which was accompanied by an insignificant increase in neutrophil diapedesis. After fMLP stimulation there was reduced neutrophil emigration in the presence of mast cell inhibitors, such as lodoxamide (1 microgram/kg) and ketotifin (microgram/kg). Our results demonstrate that both the endothelial cells and interstitial mast cells are involved in the rate of transvascular migration of leukocytes across postcapillary venules and into the interstitium.

Ischemia/reperfusion-induced granulocyte influx is a multistep process mediated by mast cells.

The influx of polymorphonuclear leukocytes (PMNs) that occurs at the onset of reperfusion of postischemic tissue is a multistep mechanism that includes initial rolling of PMNs, a progressive reduction in PMN rolling velocity, and ultimately PMN adhesion and emigration. In this study we assessed whether mast cells play a role in reperfusion-induced PMN recruitment with emphasis on PMN rolling, adhesion, and emigration. Cat small intestinal venules were visualized using intravital microscopy during control, 60 min of ischemia (intestinal blood flow 20% of control), and 60 min of reperfusion in untreated and cromolyn- (mast cell stabilizer) pretreated animals. Between 1 and 5 min of reperfusion, there was a very dramatic rise in PMN rolling which persisted for the next 60 min. In cromolyn-pretreated animals, the flux of rolling PMNs increased in the very early (5 min) reperfusion period, but by 10 min the number of rolling leukocytes was significantly reduced, an event that persisted for the next 60 min. PMNs rolled at a velocity of 40% of control values for the first 10 min of reperfusion without a change in hemodynamic values but returned toward control values by 60 min of reperfusion. An identical velocity profile was observed for the cromolyn-treated animals. In untreated animals, PMN adhesion and emigration was significantly increased throughout the reperfusion period, whereas the cromolyn-pretreated animals had reduced adhesion (60 min of reperfusion) and a reduced rate of emigration throughout the reperfusion period. Interstitial mast cells contribute significantly to the multistep recruitment (rolling, adhesion, and emigration) of PMNs from blood to postischemic tissues and raise the possibility that antiallergy agents such as cromolyn may be a useful form of therapy in the postischemic intestine.

Microvascular oxidative stress preceding leukocyte activation elicited by in vivo nitric oxide suppression.

This study was aimed to determine the mechanism by which endogenous nitric oxide suppression promotes leukocyte adhesion in vivo. The rat mesenteric microcirculation was superfused with NG-nitro-L-arginine methyl ester (L-NAME; 100 microM), and intracellular oxidant formation in several microcirculatory cellular components such as arteriolar and venular endothelium and mast cells was visually monitored by digital microfluorography assisted by carboxydichlorofluorescein (CDCF), a hydroperoxide-sensitive fluorogenic probe. Adherent leukocyte density was measured simultaneously. L-NAME induced a significant time-dependent increase in CDCF fluorescence in arteriolar and venular endothelium and mast cells followed by firm adhesion of leukocytes. L-NAME-induced CDCF elevation showed a different spatial distribution compared with that evoked by N-formylmethionyl-leucyl-phenylalanine, in which only local venular segments with adhering leukocytes exhibited CDCF fluorescence enhancement. The level of hydroperoxide formation in arterioles and venules evoked by 60-min L-NAME superfusion was equivalent to that induced by the superfusion of approximately 880 microM tert-butyl hydroperoxide for 10 min. Pretreatment with anti-intracellular adhesion molecule-1, anti-P-selectin, or anti-CD18 monoclonal antibody attenuated L-NAME-elicited venular leukocyte adhesion without abolishing CDCF fluorescence in situ. Pretreatment with desferioxamine (50 mg/kg iv; 1 h before L-NAME superfusion) significantly diminished the iron-catalyzed hydroperoxide formation in arterioles and venules, but not in interstitial mast cells, as well as subsequent venular leukocyte adhesion. These findings indicate that endogenous nitric oxide may modulate oxidative stress in mast cells, arteriolar and venular microvascular endothelium and thereby can play a crucial role in leukocyte recruitment in venules.

Temporal Sequence of Early Alterations in Rat Lung Following Thoracic X-irradiation

The temporal patterns of several responses in the lungs of groups of rats administered two different thoracic doses of X-rays (7.5 and 15 Gy) were examined during a time frame within which radiation pneumonitis develops (1-13-week period) in order to assess for potential interrelationships among the responses. Endpoints surveyed included lung gravimetric and volumetric changes, changes in lavaged alveolar macrophage (AM) numbers, interstitial accumulations of mast cells (MC), and alterations in the amounts of lavaged phospholipids (PL), protein (P), and histamine (H). Sham-irradiated rats served as controls. Early (1 week), dose-dependent increases in lavageable PL were not accompanied by increases in lung weights or lavageable P. Elevations in lavaged PL continued for at least 5 weeks after exposure. By this time, lung weights, AM numbers, MC, and lavageable P, but not lavageable H, were all substantially increased in lungs that received the 15 Gy dose, whereas these changes were not observed at the lower dose. At later times (weeks 7 and 9), the hyperpermeability response following the 15 Gy exposure became less pronounced and the PL response also subsided, while lung MC continued to further increase and lavageable H became abnormally elevated. Maximal increases in the lung's permeability status after the 15 Gy exposure was coincidental with maximal increases in AM. An association of increases in AM and lung hyperpermeability, however, was not evident as of week 13 after the 7.5 Gy dose when lung weights and lavageable P were significantly elevated. For the X-ray doses examined, our results suggest that: (1) the early PL response to X-rays is independent of, and precedes, permeability changes in the lung; (2) the time to onset of an X-irradiation-induced increase in lung permeability is dose-dependent; (3) the progressive accumulation of MC in the lung following X-irradiation is dose-dependent; (4) excessive accumulations of interstitial MC after X-irradiation do not necessarily result in an increase in free H; and (5) free H in the lung does not appear to play a prominent role in the hyperpermeable response. Additionally, in conjunction with the findings of other investigators, our study suggests that the MC response to X-rays may be a fundamental component of the fibrogenic response.

The uptake of 5-hydroxytryptamine in endothelial cells cultured from the pulmonary artery in rats. A cytochemical study.

Endothelial cells from the rat pulmonary artery were cultured and identified ultrastructurally and immunocytochemically by the presence of factor VIII antigens. Monolayers of cultured cells were treated according to the Hillarp-Falck technique and studied cytofluorimetrically to estimate intracellular serotonin (5-HT) levels. An uptake of 5-HT from the incubation medium was demonstrated at a concentration of 10(-3) M. However, after inhibition of monoamine oxidase (nalamide 5 X 10(-4) M) to prevent intracellular degradation of the amine an uptake could be demonstrated at 10(-5) M. The amine uptake into endothelial cells utilizes an active pumpmechanism, since it was effectively antagonized by either imipramine (10(-4) M) or ouabain (10(-5) M). These in vitro findings contrast earlier cytofluorimetric in vivo studies, where 5-HT uptake predominantly occurred in interstitial mast cells.