Migration Of Polymorphonuclear Granulocytes Research Articles

Inhibition of CXCR4 and CXCR7 Is Protective in Acute Peritoneal Inflammation.

Our previous studies revealed a pivotal role of the chemokine stromal cell-derived factor (SDF)-1 and its receptors CXCR4 and CXCR7 on migratory behavior of polymorphonuclear granulocytes (PMNs) in pulmonary inflammation. Thereby, the SDF-1-CXCR4/CXCR7-axis was linked with adenosine signaling. However, the role of the SDF-1 receptors CXCR4 and CXCR7 in acute inflammatory peritonitis and peritonitis-related sepsis still remained unknown. The presented study provides new insight on the mechanism of a selective inhibition of CXCR4 (AMD3100) and CXCR7 (CCX771) in two models of peritonitis and peritonitis-related sepsis by injection of zymosan and fecal solution. We observed an increased expression of SDF-1, CXCR4, and CXCR7 in peritoneal tissue and various organs during acute inflammatory peritonitis. Selective inhibition of CXCR4 and CXCR7 reduced PMN accumulation in the peritoneal fluid and infiltration of neutrophils in lung and liver tissue in both models. Both inhibitors had no anti-inflammatory effects in A2B knockout animals (A2B–/–). AMD3100 and CCX771 treatment reduced capillary leakage and increased formation of tight junctions as a marker for microvascular permeability in wild type animals. In contrast, both inhibitors failed to improve capillary leakage in A2B–/– animals, highlighting the impact of the A2B-receptor in SDF-1 mediated signaling. After inflammation, the CXCR4 and CXCR7 antagonist induced an enhanced expression of the protective A2B adenosine receptor and an increased activation of cAMP (cyclic adenosine mono phosphate) response element-binding protein (CREB), as downstream signaling pathway of A2B. The CXCR4- and CXCR7-inhibitor reduced the release of cytokines in wild type animals via decreased intracellular phosphorylation of ERK and NFκB p65. In vitro, CXCR4 and CXCR7 antagonism diminished the chemokine release of human cells and increased cellular integrity by enhancing the expression of tight junctions. These protective effects were linked with functional A2B-receptor signaling, confirming our in vivo data. In conclusion, our study revealed new protective aspects of the pharmacological modulation of the SDF-1-CXCR4/CXCR7-axis during acute peritoneal inflammation in terms of the two hallmarks PMN migration and barrier integrity. Both anti-inflammatory effects were linked with functional adenosine A2B-receptor signaling.

Sequential transmigration of polymorphonuclear cells and naive CD3+ T lymphocytes across the blood-cerebrospinal-fluid barrier in vitro following infection with Echovirus 30

Viral meningitis by non-polio enteroviruses (NPEV) is a major public health burden causing fatal outcomes especially in the younger population. Strong evidence exists that the blood-cerebrospinal-fluid (CSF) barrier (BCSFB) serves as an entry point for enterovirus and leucocytes into the central nervous system (CNS). Moreover, analysis of clinical CSF specimens of patients with a NPEV infection revealed a predominance of polymorphonuclear granulocytes (PMN) in the early phase and mononuclear cells in the later course of meningitis. By applying a functional in vitro model of the BCSFB consisting of human choroid plexus papilloma (HIBCPP) cells, we aimed to analyse the mechanisms of sequential migration of PMN and naive CD3+ T lymphocytes following infection with Echovirus 30 (EV30). EV30 infection led to increased transmigration of PMN and naive CD3+ T lymphocytes. Transmigration of PMN was significantly enhanced in the presence of naive CD3+ T lymphocytes, but not vice versa. The barrier function was not differentially altered under the respective conditions. Infection with EV30 led to an upregulation of CXCL3 and CXCL11 on the RNA-level. Additional analysis of cytokine secretion revealed relatively high concentrations of IL-8, CCL20, CXCL3, CXCL10 and M-CSF. Overall, there was a predominantly polar direction of cytokine secretion to the basolateral side. IL-7 was the only cytokine which was strongly secreted to the apical side and that was enhanced following EV30 infection in our model. In conclusion, this study highlights the role of the choroid plexus and cytokines in regulating leucocyte entry into the CNS in the context of EV30 infection.

The neurovascular unit as a selective barrier to polymorphonuclear granulocyte (PMN) infiltration into the brain after ischemic injury.

The migration of polymorphonuclear granulocytes (PMN) into the brain parenchyma and release of their abundant proteases are considered the main causes of neuronal cell death and reperfusion injury following ischemia. Yet, therapies targeting PMN egress have been largely ineffective. To address this discrepancy we investigated the temporo-spatial localization of PMNs early after transient ischemia in a murine transient middle cerebral artery occlusion (tMCAO) model and human stroke specimens. Using specific markers that distinguish PMN (Ly6G) from monocytes/macrophages (Ly6C) and that define the cellular and basement membrane boundaries of the neurovascular unit (NVU), histology and confocal microscopy revealed that virtually no PMNs entered the infarcted CNS parenchyma. Regardless of tMCAO duration, PMNs were mainly restricted to luminal surfaces or perivascular spaces of cerebral vessels. Vascular PMN accumulation showed no spatial correlation with increased vessel permeability, enhanced expression of endothelial cell adhesion molecules, platelet aggregation or release of neutrophil extracellular traps. Live cell imaging studies confirmed that oxygen and glucose deprivation followed by reoxygenation fail to induce PMN migration across a brain endothelial monolayer under flow conditions in vitro. The absence of PMN infiltration in infarcted brain tissues was corroborated in 25 human stroke specimens collected at early time points after infarction. Our observations identify the NVU rather than the brain parenchyma as the site of PMN action after CNS ischemia and suggest reappraisal of targets for therapies to reduce reperfusion injury after stroke.Electronic supplementary materialThe online version of this article (doi:10.1007/s00401-012-1076-3) contains supplementary material, which is available to authorized users.

The cellular cancer resistance of the SR/CR mouse

The SR/CR mouse phenotype, first described in 1999 in BALB/c and later bred into C57BL/6 mice, is resistant to cancer formation following high doses of cancer cells administered intraperitoneally. The tumor cell targeting and destruction mechanisms have not been identified. By fluorescence-activated cell sorting analysis, the immune response of SR/CR mice after intraperitoneal injection of cancer cells was investigated and compared with parent strain mice. A massive influx of leukocytes into the peritoneal cavity was found. A large fraction of these leukocytes were polymorphonuclear granulocytes, macrophages and natural killer cells. A relative decrease in influx of B-cells compared with controls was demonstrated. Increased proportions of leukocytes belonging to the innate immune system were also demonstrated in splenocytes of SR/CR mice. Cytospins of peritoneal fluid from SR/CR mice after cancer cell injection showed formations of immune cells morphologically resembling polymorphonuclear granulocytes and macrophages adjoining the cancer cells. The results point to the potential involvement of innate immune cells in cancer immunology. Our data support migration of polymorphonuclear granulocytes, macrophages and NK cells into the peritoneum of the SR/CR mouse in response to intraperitoneal injection of S180 cancer cells. The cell composition of spleens of SR/CR mice reflected the differential regulation of the innate immune cells in peritoneal exudates. Both peritoneal exudates and the spleens of SR/CR mice contained decreased proportions of B-cells compared with BALB/c and C57BL/6 mice. We reproduce important aspects of previous published data and further extend them by showing differentially regulated populations of splenocytes including B-lymphocytes in SR/CR mice compared with parent strain controls. Importantly, this differentially regulated immune response of SR/CR mice could not be found in response to challenge with the lymphoma cell line EL-4.

Rabbit polymorphonuclear granulocyte function during ethanol administration--migration and oxidative responses in a joint with immune complex synovitis.

Functional impairments of polymorphonuclear granulocytes (PMN) are believed to contribute to hampered inflammation and host defence in alcoholics. We studied effects of i.v. ethanol administration on PMN responses in rabbits during induction of a knee-joint synovitis. The synovitis conferred systemic effects, since chemiluminescent responses of peripheral blood PMN to opsonized zymosan and phorpbol myristate acetate (PMA) increased 6.4- and 17.9-fold, respectively. Chemiluminescent responses of synovial PMN were further amplified. This up-regulation was reduced to 33% in rabbits treated with ethanol when opsonized zymosan was used as the PMN stimulus; in contrast, PMA responses were unaffected. The appearance and migration of PMN to the synovitis joint were normal despite a blood ethanol concentration of 0.5%. Thus, ethanol impaired release of oxygen metabolites from PMN, but not the delivery of cells at an inflammatory site.

Cell Adhesion and Migration Properties of β2-Integrin Negative Polymorphonuclear Granulocytes on Defined Extracellular Matrix Molecules: RELEVANCE FOR LEUKOCYTE EXTRAVASATION

Regulated adhesion of leukocytes to the extracellular matrix is essential for transmigration of blood vessels and subsequent migration into the stroma of inflamed tissues. Although beta(2)-integrins play an indisputable role in adhesion of polymorphonuclear granulocytes (PMN) to endothelium, we show here that beta(1)- and beta(3)-integrins but not beta(2)-integrin are essential for the adhesion to and migration on extracellular matrix molecules of the endothelial cell basement membrane and subjacent interstitial matrix. Mouse wild type and beta(2)-integrin null PMN and the progranulocytic cell line 32DC13 were employed in in vitro adhesion and migration assays using extracellular matrix molecules expressed at sites of extravasation in vivo, in particular the endothelial cell laminins 8 and 10. Wild type and beta(2)-integrin null PMN showed the same pattern of ECM binding, indicating that beta(2)-integrins do not mediate specific adhesion of PMN to the extracellular matrix molecules tested; binding was observed to the interstitial matrix molecules, fibronectin and vitronectin, via integrins alpha(5)beta(1) and alpha(v)beta(3), respectively; to laminin 10 via alpha(6)beta(1); but not to laminins 1, 2, and 8, collagen type I and IV, perlecan, or tenascin-C. PMN binding to laminins 1, 2, and 8 could not be induced despite surface expression of functionally active integrin alpha(6)beta(1), a major laminin receptor, demonstrating that expression of alpha(6)beta(1) alone is insufficient for ligand binding and suggesting the involvement of accessory factors. Nevertheless, laminins 1, 8, and 10 supported PMN migration, indicating that differential cellular signaling via laminins is independent of the extent of adhesion. The data demonstrate that adhesive and nonadhesive interactions with components of the endothelial cell basement membrane and subjacent interstitium play decisive roles in controlling PMN movement into sites of inflammation and illustrate that beta(2)-integrins are not essential for such interactions.

Influence of antiseptic agents on interleukin 8 release and transmigration of polymorphonuclear granulocytes in an in vitro model of peritonitis

Abstract Background The effect of antiseptic agents on peritoneal cells is ill defined. The influence of taurolidine (TAU) and polyhexamide (HEX) was investigated in an in vitro model. Methods Human umbilical vein endothelial cells (HUVECs) and human peritoneal mesothelial cells (HPMCs) were laid on collagen-coated filter inserts (HUVECs on the bottom, HPMCs on the top), thus representing a two-chamber peritoneal model. When confluence was reached, HPMCs were stimulated with 0·5 ml tumour necrosis factor (TNF) α 10 μg ml−1 for 4 h. Afterwards 0·5 ml TAU (1 and 2 per cent) or 0·5 ml HEX (0·1 and 0·2 per cent) solutions were added to the upper compartment. After 1 h polymorphonuclear granulocytes (PMNs) (105 ml−1) were added to the lower compartment. After 2 and 6 h aliquots were taken from both compartments, transmigrated PMNs were counted and interleukin (IL) 8 concentrations were measured. Controls were either TNF-α-stimulated HPMCs or stimulated HPMCs where culture medium had been substituted for TNF-α. Significance of differences was assessed by analysis of variance with Bonferroni corrections. Correlations were calculated by linear regression analysis. Results Stimulation with TNF-α led to a time-dependent increase in PMN transmigration. IL-8 secretion into the apical compartment increased time dependently, resulting in a gradient between the two chambers. After substitution of the stimulus by culture medium, significantly less IL-8 was measured in both compartments. PMN transmigration was almost absent. Addition of HEX resulted in an initial increase in IL-8 levels comparable to TNF controls without further changes. A concentration-dependent decrease in IL-8 gradient was associated with reduced transmigration. The IL-8 gradient between the upper and lower chambers correlated significantly with PMN transmigration (r = 0·8205, P < 0·0001). Conclusion The decrease in IL-8 gradients by HEX and the diminished IL-8 response after addition of TAU may reflect either anti-inflammatory effects or cellular damage. Both antiseptic solutions reduced PMN migration, irrespective of continuous stimulation in this model.

Plant-derived acetophenones with antiasthmatic and anti-inflammatory properties: inhibitory effects on chemotaxis, right angle light scatter and actin polymerization of polymorphonuclear granulocytes.

3-Methoxy- and 2-hydroxy-3-methyl substituted 4-hydroxyacetophenones and some of the corresponding 4-O-glucosides revealed inhibitory activity on LTB4-, fMLP- and PAF-induced chemotaxis of polymorphonuclear granulocytes (PMN). Blocking of PMN myeloperoxidase (MPO), which is required for the intracellular activation of acetophenones, caused a loss of activity. By in vitro activation of apocynin (1) with human MPO and H2O2 and subsequent chromatographic purification an apocynin-free fraction was obtained, which contained the major metabolite. The fraction showed strong anti-chemotactic activity, which was largely preserved despite of MPO-blockade. The influences on right angle light scatter and actin polymerization in PMN demonstrate the inhibitory effects of acetophenones on the cytoskeletal rearrangement after stimulation with fMLP. Acetosyringenin (2) was shown to reduce mastoparan-induced PMN migration. The inhibition of receptor independent motility of PMN suggests that the active metabolites of acetophenones may interfere with the post receptor signalling.

Changes of polymorphonuclear granulocyte migration and lymphocyte proliferative responses in elite runners undergoing intense exercise

The immunoregulatory effects of intense exercise were examined measuring polymorphonuclear granulocyte (PMN) migration under agarose, lymphocyte proliferative responses after mitogen stimulation (phytohemagglutinin P, concanavalin A and pokeweed mitogen) and complement activation (C3 conversion) in the peripheral blood of elite runners undergoing maximal exertion. Eleven elite middle‐ and long‐distance runners were tested in a 5‐km race on a running track. PMN migration activity towards autologous serum and FMLP as chemoattractants both increased after intense physical activity. The lymphocytes showed increased proliferative responses to the mitogen stimulation 2 h after the run compared with pre‐exercise values, and normalized activity after 24 h. The total number of lymphocytes in the peripheral blood markedly decreased 2 h after the run and the number of leukocytes increased. A tendency to a decrease in complement C3 conversion‐was observed after the run, but within the estimated normal area. Our findings suggest that intense physical activity influences the cellular immune system.

Changes in PMN leukocyte migration activity and complement C3d levels in RA patients with high disease activity during steroid treatment.

We have investigated chemotaxis of polymorphonuclear granulocytes (PMN) from 7 well characterized RA patients with intense disease activity, before and after steroid treatment. Increased spontaneous and directed granulocyte migration was observed in 6 out of 7 patients before steroid treatment. In 5 patients these parameters were reduced after treatment. In contrast to this, decreased chemotaxis of polymorphonuclear granulocytes (PMN) from patients with Rheumatoid Arthritis (RA), have been reported earlier. This has been suggested as a partial explanation of the increased morbidity of these patients. Plasma levels of Complement C3d were initially increased in all patients and were slightly reduced following treatment. PMN migration in highly active RA cases is increased and responds markedly to steroid treatment, in contrast to the complement activation. The mainly unaffected, increased levels of plasma C3d might, in part, explain the early recurrence of symptoms often seen when steroid medication is withdrawn. Our results contrast with earlier findings and need to be confirmed in further studies.

Effects of two macrolide antibiotics on human leukocyte membrane receptors and functions.

Polymorphonuclear granulocytes (PMNs) and lymphocytes from healthy persons were incubated in varying concentrations of erythromycin and RU 28965, a new macrolide antibiotic. Incubation in erythromycin - even in high dilutions - caused a significant increase in the percentage of PMNs bearing receptors for the Fc portion of IgG (Fc gamma R) and for C3b (C3bR) as measured by rosette formation with EA (erythrocyte-antibody) and EAC (erythrocyte-antibody-complement) indicator cells. This effect could not be removed by extended washing of the cells. Incubation in RU 28965 had a similar effect, except for a decrease in EA and EAC rosetting cells at high concentrations (200 mg/l). Phagocytosis, as measured by chemiluminescence, and random migration of PMNs were unaffected by erythromycin. Chemotaxis under agarose was decreased after incubation in erythromycin or RU 28965. Erythromycin incubation increased the percentage of lymphocytes bearing receptors for sheep erythrocytes (E), but had no effect on the proportion of lymphocytes rosetting with EA or EAC, or on lymphocyte responses to mitogens PHA, conA, or PWM.

Granulocytes in bronchial lavage fluid after major vascular surgery.

Increased numbers of polymorphonuclear granulocytes (PMN) in the airways, as measured by PMN content in bronchial lavage fluid (P less than 0.01), were found 3 h postoperatively in ten patients undergoing surgery for lumbar aortic aneurysms. An increase in plasma levels of the complement split product C3dg from 6 (0-19) AU/ml preoperatively to 20 (13-50) AU/ml 3 h after surgery (P less than 0.01), indicates an activation of the complement cascade. These changes were not accompanied by increased elastase activity in the bronchial lavage fluid or by major changes in pulmonary blood gas exchange or vascular resistance, indicating that massive PMN activation, analogous to that proposed in adult respiratory distress syndrome (ARDS) had not taken place. In conclusion, complement system activation and migration of PMN into the airways, as seen in connection with major vascular surgery, does not seem to contribute to ARDS-type pulmonary dysfunction.

The effect of pregnancy on functions of inflammatory cells in healthy women and in patients with rheumatic disease.

Chemiluminescence (CL) after zymosan stimulated phagocytosis of polymorphonuclear granulocytes (PMN) and mononuclear cells (MNC), random migration of PMN and intra- and extracellular activities of nine lysosomal enzymes were assessed serially in 8 healthy women and 10 women with rheumatic disease during and after pregnancy. A gestational increase of lysosomal enzymes in serum and enhancement of PMN random migration was observed in all women. Significant differences between healthy and rheumatic women were found for CL of phagocytic cells. In healthy women, CL of PMN was slightly enhanced, while it remained unchanged in MNC during pregnancy. In patients, CL of PMN was markedly suppressed, while MNC CL increased during gestation. An inverse relationship between CL and intracellular enzyme activities was noted. Thus, the presence of an inflammatory state seemed to influence the gestational behavior of phagocytic cells.

Acute synovitis induced by preformed immune complexes.

The morphology of acute immune complex (IC) elicited synovitis in rabbit knee joints was studied, as well as IC-induced leukocyte activation in vivo and in vitro. Acute synovitis was induced by intra-articular injection of in vitro preformed, complement activating bovine serum albumin (BSA)-anti-BSA IC. Within 30 min, migration of polymorphonuclear granulocytes (PMNGs) was observed. Scanning electron microscopy showed that adhering, apparently activated leukocytes were attached to the synovial lining, often forming clusters. Phagocytosis of IC was evident, as immunoglobulins were detected within the leukocyte cytoplasm by the direct immunofluorescence technique. At the peak accumulation of PMNGs, focal erosions of the synovial lining were observed. Later, monocytes and macrophages appeared and degenerated PMNGs were found, sometimes within the cytoplasm of large macrophages. Chemiluminescence experiments showed a maximum in vitro activation of leukocytes by complement activating IC formed near optimal precipitation proportions and in slight antigen excess, whereas IC in large antigen excess gave a smaller and later response.

The effect of auranofin on polymorphonuclear granulocytes.

The effects of auranofin on the function of neutrophil polymorphonuclear granulocytes (PMN) have been studied in vitro and in vivo. Preincubation of human PMN with auranofin (1-4 micrograms/ml) increased their adherence to nylon fibres and f-met-leu-phe-(fMLP) induced aggregation. PMN migration, phagocytosis, bactericidal capacity and phagocytosis-associated enzyme release were all significantly inhibited by auranofin in a dose-dependent way. Enzyme release stimulated by f-MLP, chemoluminescence and the release of superoxide anions all showed a biphasic response to preincubation with auranofin. They showed an increase at low concentrations and inhibition at high concentrations. In studies of 3H-fMLP binding auranofin did not affect receptor numbers but increased binding affinity. Auranofin at higher concentrations decreased phorbolmyristate acetate and fMLP induced changes in surface charge and membrane potential. In vivo, auranofin administered to rats, did not prevent either the neutropenia induced by zymosan-activated serum or the corresponding rise in plasma lactoferrin levels. PMNs from six rheumatoid arthritis patients treated with auranofin (6 mg/day) for 23 weeks showed changes in bactericidal activity, chemotaxis and chemiluminescence independent of the clinical response. Enzyme release, however, was reduced in PMNs from clinical responders and showed no change in non-responders.