Blockade of proteinase-activated receptor 4 inhibits neutrophil recruitment in experimental inflammation in mice

Objective and design

The activation of proteinase-activated receptors (PARs) has been implicated in the development of important hallmarks of inflammation, including in vivo leukocyte recruitment; however, its role in the regulation of leukocyte migration in response to inflammatory stimuli has not been elucidated until now. Here, we examined the effects of the PAR4 antagonist YPGKF-NH 2 (tcY-NH₂) on neutrophil recruitment in experimentally induced inflammation.

Methods

BALB/c mice were intrapleurally injected with tcY-NH₂ (40 ng/kg) prior to intrapleural injection of carrageenan (Cg) or neutrophil chemoattractant CXCL8; the number of infiltrating neutrophils was evaluated after 4 h, and KC production was assessed at different times after Cg injection. Neutrophil adhesion and rolling cells were studied using a brain circulation preparation 4 h after the Cg or CXCL8 challenge in tcY-NH₂-treated mice.

Results

PAR4 blockade inhibited CXCL8- and Cg-induced neutrophil migration into the pleural cavity of BALB/c mice and reduced neutrophil rolling and adherence. Surprisingly, PAR4 blockade increased the level of KC in response to carrageenan.

Conclusion

These results demonstrated that PAR4 blockade impairs neutrophil migration in vivo, suggesting that PAR4 plays an important role in the regulation of inflammation, at least in part because of its ability to inhibit the actions of the neutrophil chemoattractant CXCL8.     

Simvastatin abrogates inflamed neutrophil adhesive properties, in association with the inhibition of Mac-1 integrin expression and modulation of Rho kinase activity

Objectives

Leukocytes play a primary role in vascular inflammation, and thus an understanding of the pathways involved in the activation of these cells and means to inhibit their consequent adhesion to the vessel wall is of significant interest. This study aimed to determine whether statins have a direct effect upon neutrophil adhesive properties under inflammatory conditions.

Methods

Neutrophils from healthy individuals were subjected to adhesion assays (with fibronectin as ligand) and flow cytometry.

Results

In the presence of a TNF-α inflammatory stimulus, neutrophils displayed a rapid and substantial enhancement in their adhesive properties that was abrogated by preincubation of cells with simvastatin. Neutrophil surface expression of the Mac-1 integrin subunit, CD11b, was augmented by TNF-α, and this increased expression was also inhibited by simvastatin. TNF-α also induced neutrophil LFA-1 and Mac-1 activation, but this activation was not blocked by simvastatin. Interestingly, while addition of the isoprenoids, geranygerayl pyrophosphate and farnesyl pyrophosphate, to cells did not alter the effect of simvastatin on TNF-α-stimulated adhesion, concurrent incubation of cells with the Rho kinase (ROCK) inhibitor reversed the effects of simvastatin on neutrophil adhesion and CD11b expression.

Conclusion

Simvastatin appears to have direct anti-inflammatory effects in neutrophils that may be mediated by modulation of ROCK activity.     

Fractionated irradiations lead to chronic allergic airway inflammation through increasing the influx of macrophages

Objective

In our previous study, repeated irradiations showed persistent depression of immune response, especially Th1-related immune response. Here, we hypothesized and determined that irradiation may exacerbate development of allergic airway inflammation.

Methods

C57BL/6 mice were irradiated repeatedly at 1 Gy or 0.5 Gy. At 6 months after irradiation, mice were sensitized and challenged short-term with OVA. Antigen-specific immunoglobulins, the percentages of inflammatory cells, chemokine expression, cytokine levels, and collagen deposition were tested.

Results

In irradiated mice, IgG2a in serum was lower when compared with that of control mice, while IgG1 was significantly higher. Interestingly, the percentages of macrophages in bronchoalveolar lavage fluid (BALF) and the lung of irradiated mice were significantly higher. Conversely, the percentages of neutrophil were significantly lower in BALF of irradiated mice. In the lung of irradiated mice, MCP-1 and IP-10 for attraction of macrophages showed the higher expression level, but KC expression for neutrophils showed no difference. Next, TGF-β1 and IL-17A in BALF were higher in irradiated mice. In addition, phosphorylated-Smad2/3 was increased in irradiated mice. Finally, the deposition of collagen was increased in irradiated mice.

Conclusion

Our study showed that fractionated irradiation lead to the chronic allergic airway inflammation through increasing the influx of macrophages and active TGF-β levels.     

Role of main neuroendocrine pathways activated by swim stress on mast cell-dependent peritoneal TNF production after LPS administration in mice

Objective and design

To characterize the effects of swim stress on the early mast cell (MC)-dependent peritoneal production of TNF in response to lipopolysaccharide (LPS) administration in mice, identifying the neuroendocrine mediators involved.

Subjects

Ten to twelve-week-old Swiss Webster, C57BL/6 J or c-Kit ^Wsh/Wsh mice were used.

Treatment

Animals were intraperitoneally challenged with LPS at different times after forced swimming (FS) and peak TNF production was determined in peritoneal washes at optimal time after LPS administration. Selective blockage of main neuroendocrine pathways was performed before swim stress. Methods: TNF concentrations were determined by ELISA.

Results

FS provoked an immediate and transient inhibition of LPS-elicited, MC-dependent TNF accumulation in peritoneum, which lasted around 30 min. Suppresive effects of FS were absent on MC-deficient c-Kit ^Wsh/Wsh mice but were recovered after reconstitution with MC. Adrenalectomy or DSP4 administration increased basal ip TNF levels and enhanced LPS-induced TNF release without any effect on stress-induced inhibitory effects, mifepristone did not produce any change on stress-induced inhibition, whereas mecamylamine administration increased basals and attenuated stress effects.

Conclusions

Swim stress transiently inhibits the canonical MC-dependent response of TNF production in response to LPS in murine peritoneal cavity with the main participation of the cholinergic anti-inflammatory reflex.     

Main roads to melanoma

Background

Since the V617F mutation in JAK2 may not be the initiating event in myeloprofilerative disorders (MPDs) we compared molecular changes in neutrophils from patients with polycythemia vera (PV) and essential thrombocythosis (ET), to neutrophils stimulated by G-CSF administration and to normal unstimulated neutrophils

Methods

A gene expression oligonucleotide microarray with more than 35,000 probes and a microRNA (miR) expression array with 827 probes were used to assess neutrophils from 6 MPD patients; 4 with PV and 2 with ET, 5 healthy subjects and 6 healthy subjects given G-CSF. In addition, neutrophil antigen expression was analyzed by flow cytometry and 64 serum protein levels were analyzed by ELISA.

Results

Gene expression profiles of neutrophils from the MPD patients were similar but distinct from those of healthy subjects, either unstimulated or G-CSF-mobilized. The differentially expressed genes in MPD neutrophils were more likely to be in pathways involved with inflammation while those of G-CSF-mobilized neutrophils were more likely to belong to metabolic pathways. In MPD neutrophils the expression of CCR1 was increased and that of several NF-κB pathway genes were decreased. MicroRNA miR-133a and miR-1 in MPD neutrophils were down-regulated the most. Levels of 11 serum proteins were increased in MPD patients including MMP-10, MMP-13, VCAM, P-selectin, PDGF-BB and a CCR1 ligand, MIP-1α.

Conclusion

These studies showed differential expression of genes particularly involved in inflammatory pathways including the NF-κB pathway and down-regulation of miR-133a and miR-1. These two microRNAs have been previous associated with certain cancers as well as the regulation of hyperthrophy of cardiac and skeletal muscle cells. These changes may contribute to the clinical manifestations of the MPDs.     

The Effects of Bruton Tyrosine Kinase Inhibition on Chemotaxis and Superoxide Generation in Human Neutrophils

Purpose

The role of the Bruton tyrosine kinase (Btk) protein in neutrophil function has been evaluated using neutrophils from healthy volunteers after incubation with a Btk inhibitor, leflunomide metabolite analog (LFM-A13), suggesting an important role for Btk in neutrophil function. We sought to determine the role of Btk protein on neutrophil superoxide generation and chemotaxis stimulated by N-formyl-methionine-leucine-phenylalanine (fMLP).

Methods

Chemotaxis was assayed on agarose gel and superoxide generation by cytochrome C reduction. The affects of LFM-A13 on chemotaxis and superoxide generation in unstimulated and fMLP stimulated neutrophils were studied in Btk deficient neutrophils from XLA patients compared with matched controls analyzed simultaneously.

Results

Chemotaxis and stimulated superoxide production were similar in the normal and Btk deficient neutrophils and were similarly inhibited by LFM-A13. In one patient, LFMA13 had no effect on superoxide generation in Btk deficient neutrophils up to a concentration of 25 microM, while inhibited superoxide production by control neutrophils.

Conclusions

Our results suggest that Btk does not have a specific role in neutrophil fMLP-stimulated superoxide generation and chemotaxis since these activities were similarly inhibited by LFM-A13 in Btk deficient and normal neutrophils. The lack of superoxide generation following Btk inhibition by LFM-A13 in Btk deficient neutrophils from one patient may suggest some heterogeneity in the role of Btk in fMLP induced neutrophil superoxide generation.     

Dynamic changes of peritoneal macrophages and subpopulations during ulcerative colitis to metastasis of colorectal carcinoma in a mouse model

Objective and design

Patients with ulcerative colitis have increased risk of colorectal carcinoma, but little is known about how peritoneal macrophages are involved in ulcerative colitis-associated carcinogenesis. We investigated the alteration of peritoneal macrophages and M1/M2 subpopulations during ulcerative colitis-associated carcinogenesis.

Materials and methods

Expression and functional changes in peritoneal macrophages and M1/M2 subpopulations were investigated by histopathology, flow cytometry, immunofluorescence, cytokines expression by ELISA and QRT-PCR in an azoxymethane (AOM)- and dextran sodium sulfate (DSS)-induced chemical colitis-associated carcinoma mouse model using male Crj:CD-1 (ICR) mice.

Results

Striking evidence observed in histopathology, flow cytometry, cytokine detection, and gene expression analysis all revealed that inflammation-associated cytokines (IL-1β, IL-10, IL-12, IL-6, TNF-α) and migration/invasion-associated factors (G-CSF, GM-CSF, CXCR4, VEGF, TGF-β, ICAM-1) induced by peritoneal M2 macrophages increased significantly during the progression from inflammatory hyperplasia to carcinoma and metastasis. Similar functional changes occurred during peritoneal metastasis in M1 macrophages without changed polarization.

Conclusions

These results suggested that peritoneal M2 macrophages played a critical role in ulcerative colitis-associated carcinogenesis, including unbalanced pro-inflammatory and anti-inflammatory axis and enhanced expression of migration/invasion-associated factors. Furthermore, functional changes of M1 macrophages occurred without changed polarization during carcinogenesis and metastasis.     

Different inflammatory stimuli in the footpad of mice influence the kinetics of resident peritoneal cells

Objectives

Evidence from the literature that inflammation is a systemic biological phenomenon prompted us to investigate whether inoculation of different irritants to the footpad of mice might influence the kinetics of resident peritoneal cells.

Methods

Mice were inoculated in the footpad at different time intervals with Mycobacterium bovis bacillus Calmette-Guerin (BCG), Ehrlich ascitic tumor cells or lipopolysaccharide (LPS), and resident peritoneal cells were analyzed by flow cytometry.

Results

The results indicate that different stimuli induced different responses in resident peritoneal cells. FoxP3 positive regulatory T cells increased drastically in number after BCG inoculation. Conversely, tumor cell inoculation induced a decrease in FoxP3-positive T cells in the peritoneal cavity, although this effect was not statistically significant. Results also show that cells from the paw migrate to the popliteal lymph node and to the peritoneal cavity. Yet, there are cells in the peritoneal cavity that migrate to the popliteal lymph node.

Conclusion

These data show that cells from the peritoneal cavity are influenced by pathologies in remote regions of the animal. How this novel phenomenon influences overall immune responses, courses of infection and tumor growth are open to further investigation.     

Up-regulation of growth factor independence 1 in endotoxin tolerant macrophages with low secretion of TNF-α and IL-6

Objective

Endotoxin tolerance refers to a low response to lipopolysaccharide (LPS). We hypothesized that growth factor independence 1 (Gfi1) involves in the endotoxin tolerance in macrophages.

Methods

Endotoxin tolerance was induced in the RAW264.7 cell line and thioglycolate-elicited murine peritoneal macrophages by incubation with 100 ng/ml LPS for 20 h. Macrophages without the pretreatment were set as control. Both endotoxin tolerant and control cells were then stimulated with 1,000 ng/ml LPS for indicated period of incubation. Gfi1 mRNA expression and protein production were investigated by real-time PCR and Western blotting, respectively. ELISA was performed to quantify the secretion of TNF-α and IL-6.

Result

Compared with non-endotoxin tolerant macrophages, endotoxin tolerant cells secreted a lower amount of TNF-α and IL-6. The mRNA expression of Gfi1 in endotoxin tolerant macrophages was higher than that of control in both RAW264.7 cells and thioglycolate-elicited murine peritoneal macrophages. The protein production was accordingly up-regulated in endotoxin tolerant RAW264.7 cells.

Conclusion

In in vitro endotoxin tolerant macrophages, the expression of Gfi1 mRNA and protein were up-regulated after high dose LPS stimulation, accompanied with a blunted TNF-α and IL-6 secretion. Gfi1 might participate in the mechanism of endotoxin tolerance.     

A role for DNA-dependent activator of interferon regulatory factor in the recognition of herpes simplex virus type 1 by glial cells

Background

Subarachnoid haemorrhage (SAH) elicits rapid pathological changes in the structure and function of parenchymal vessels (≤ 100 μm). The role of neutrophils in these changes has not been determined. This study investigates the role of neutrophils in early microvascular changes after SAH

Method

Rats were either untreated, treated with vinblastine or anti-polymorphonuclear (PMN) serum, which depletes neutrophils, or treated with pyrrolidine dithiocarbamate (PDTC), which limits neutrophil activity. SAH was induced by endovascular perforation. Neutrophil infiltration and the integrity of vascular endothelium and basement membrane were assessed immunohistochemically. Vascular collagenase activity was assessed by in situ zymography.

Results

Vinblastine and anti-PMN serum reduced post-SAH accumulation of neutrophils in cerebral vessels and in brain parenchyma. PDTC increased the neutrophil accumulation in cerebral vessels and decreased accumulation in brain parenchyma. In addition, each of the three agents decreased vascular collagenase activity and post-SAH loss of vascular endothelial and basement membrane immunostaining.

Conclusions

Our results implicate neutrophils in early microvascular injury after SAH and indicate that treatments which reduce neutrophil activity can be beneficial in limiting microvascular injury and increasing survival after SAH.     

Balanced and unbalanced solutions modulate the release of Matrix Metalloproteinase-9 (MMP-9) from neutrophils in response to inflammatory stimuli: an in vitro study

Objectives and design

We investigated the effect of balanced (BS) and unbalanced (UBS) solutions in the absence or presence of hydroxyethyl starch (HES) on neutrophil functionality, evaluating the release of matrix metalloproteinase (MMP)-9, myeloperoxidase (MPO), and MMP-8.

Materials and methods

Neutrophils were isolated by gradient centrifugation and dextran sedimentation and incubated in BS or UBS without or with HES, in the absence or presence of Interleukin-8 (IL-8) or Lipopolysaccharide (LPS). MMP-9, MPO, and MMP-8 were assayed by commercially available ELISA kits.

Results

There was not any influence of volume replacement solutions on the release of the enzymes from resting neutrophils. After IL-8 stimulation, the release of MMP-9 was higher in BS than in UBS or RPMI-1640, whereas HES enhanced its release regardless of the composition. After LPS stimulation, the release of MMP-9 was higher in both UBS and BS than RPMI-1640, but HES brought its release back to physiological conditions. No difference was found in the release of MPO and MMP-8 after stimulation with IL-8 or LPS.

Conclusion

Volume replacement solutions might have an impact on the release of MMP-9 depending on the inflammatory milieu, suggesting that the use of balanced or unbalanced solutions is not a neutral choice.     

Sputum neutrophil count after bronchial allergen challenge is related to peripheral blood neutrophil chemotaxis in asthma patients

Objective

The aim of this study was to estimate relations between sputum neutrophilia and the chemotactic activity of peripheral blood neutrophils after the bronchial allergen challenge in asthma patients.

Materials and methods

Fifteen patients with allergic asthma (AA), 13 patients with allergic rhinitis (AR), all sensitized to Dermatophagoides pteronyssinus, and 8 healthy subjects (HS) underwent bronchial challenge with D. pteronyssinus. Sputum and peripheral blood collection were performed 24 h before, 7 and 24 h after the bronchial challenge. Cell counts were determined by the May-Grünwald-Giemsa method. Neutrophil chemotaxis was analyzed by a flow cytometer; IL-8 levels were measured by ELISA.

Results

Sputum neutrophil count and peripheral blood neutrophil chemotaxis of patients with AA were greater 7 and 24 h after the challenge compared with the baseline values and patients with AR and HS (P < 0.05). Moreover, a significant correlation was found between the neutrophil count in sputum and IL-8 levels, and the chemotactic activity of peripheral blood neutrophils 24 h after the bronchial challenge only the patients with AA (P < 0.05).

Conclusions

Increased sputum neutrophil count was found to be associated with an enhanced chemotactic activity of peripheral blood neutrophils during allergen-induced late-phase airway inflammation in patients with allergic asthma.     

Discriminators of mouse bladder response to intravesical Bacillus Calmette-Guerin (BCG)

Background

Intravesical Bacillus Calmette-Guerin (BCG) is an effective treatment for bladder superficial carcinoma and it is being tested in interstitial cystitis patients, but its precise mechanism of action remains poorly understood. It is not clear whether BCG induces the release of a unique set of cytokines apart from its pro-inflammatory effects. Therefore, we quantified bladder inflammatory responses and alterations in urinary cytokine protein induced by intravesical BCG and compared the results to non-specific pro-inflammatory stimuli (LPS and TNF-α). We went further to determine whether BCG treatment alters cytokine gene expression in the urinary bladder.

Methods

C57BL/6 female mice received four weekly instillations of BCG, LPS, or TNF-α. Morphometric analyses were conducted in bladders isolated from all groups and urine was collected for multiplex analysis of 18 cytokines. In addition, chromatin immune precipitation combined with real-time polymerase chain reaction assay (CHIP/Q-PCR) was used to test whether intravesical BCG would alter bladder cytokine gene expression.

Results

Acute BCG instillation induced edema which was progressively replaced by an inflammatory infiltrate, composed primarily of neutrophils, in response to weekly administrations. Our morphological analysis suggests that these polymorphonuclear neutrophils are of prime importance for the bladder responses to BCG. Overall, the inflammation induced by BCG was higher than LPS or TNF-α treatment but the major difference observed was the unique granuloma formation in response to BCG. Among the cytokines measured, this study highlighted the importance of IL-1β, IL-2, IL-3, IL-4, IL-6, IL-10, IL-17, GM-CSF, KC, and Rantes as discriminators between generalized inflammation and BCG-specific inflammatory responses. CHIP/Q-PCR indicates that acute BCG instillation induced an up-regulation of IL-17A, IL-17B, and IL-17RA, whereas chronic BCG induced IL-17B, IL-17RA, and IL-17RB.

Conclusion

To the best of our knowledge, the present work is the first to report that BCG induces an increase in the IL-17 family genes. In addition, BCG induces a unique type of persisting bladder inflammation different from TNF-α, LPS, and, most likely, other classical pro-inflammatorystimuli.     

Quercetin and kaempferol suppress immunoglobulin E-mediated allergic inflammation in RBL-2H3 and Caco-2 cells

Objective

We investigated the inhibitory effects of quercetin and kaempferol treatment on the suppression of immunoglobulin E (IgE)-mediated allergic responses in relation to intestinal epithelium barrier function in RBL-2H3 and Caco-2 cells.

Methods

RBL-2H3 cells as a model of intestinal mucosa mast cells were treated with flavonols followed by IgE-anti-dinitrophenyl sensitization. The extent of degranulation and the release of pro-inflammatory cytokines were measured. Caco-2 cells were stimulated with interleukin (IL)-4 or IgE-allergen with or without flavonol pretreatment and changes in the expression of CD23 mRNA and mitogen-activated protein kinase (MAPK), and chemokine release were determined.

Results

Flavonols inhibited the secretion of allergic mediators in RBL-2H3 cells and suppressed the CD23 mRNA expression and p38 MAPK activation in IL-4 stimulated Caco-2 cells. Flavonols also suppressed IgE-OVA induced extra signal-regulated protein kinase (ERK) activation and chemokine release.

Conclusions

Quercetin and kaempferol effectively suppressed the development of IgE-mediated allergic inflammation of intestinal cell models.     

CXCL1/KC and CXCL5/LIX are selectively produced by corneal fibroblasts and mediate neutrophil infiltration to the corneal stroma in LPS keratitis

The severity of corneal inflammation depends on the activity of infiltrating neutrophils responding to chemotactic factors such as CXC chemokines. This study examines the relative contribution of CXCL1/keratinocyte-derived chemokine (KC), CXCL2/monocyte-inhibitory protein-2 (MIP-2), and CXCL5/LPS-induced chemokine (LIX) in neutrophil recruitment to the corneal stroma during LPS keratitis, where neutrophils infiltrate the corneal stroma at 6 h after LPS injection and peak at 24 h. Consistent with this timeframe, KC was detected after 3 h, reached peak levels at 24 h, and decreased thereafter. In contrast, LIX production was not detected until 8 h after injection and peaked at 24 h. MIP-2 was detected at 3 h but did not reach the levels of KC and LIX. Cell types associated with corneal inflammation produced markedly different chemokines in vitro: Murine corneal fibroblasts (MK/T-1) produced LIX and KC in response to LPS but did not produce MIP-2, whereas peritoneal macrophages and neutrophils produced MIP-2 and KC but did not produce LIX. To determine the role of these chemokines in neutrophil recruitment to the cornea, anti-LIX, anti-KC, or anti-MIP-2 was injected into the corneal stroma of enhanced GFP chimeric mice prior to LPS, and total cell and neutrophil infiltration was examined. Antibody to LIX and KC, injected individually or in combination, significantly inhibited neutrophil recruitment to the cornea, whereas anti-MIP-2 had no inhibitory effect. Together, these findings demonstrate cell-specific production of CXC chemokines and show that LIX and KC mediate neutrophil recruitment into the cornea during LPS keratitis.     

Characterization of lipopolysaccharide-stimulated cytokine expression in macrophages and monocytes

Objective

In vitro cell culture models are widely used in inflammation research; however, information regarding the time- and dose-dependency of inflammatory responses toward LPS in these cell lines is scattered in the literature.

Material

J774A.1 mouse macrophage and THP-1 human monocyte cell lines.

Treatment

J774A.1 and THP-1 cells were treated with 0–500?ng/mL lipopolysaccharide for 0–24?h.

Methods

SRB and BCA tests were used to measure total protein. Real-time PCR was used to determine gene expression levels, and ELISA was used to assess the protein levels. One-way ANOVA and Tukey’s Honestly Significant Difference test were used to test the significance levels.

Results

In J774A.1 and THP-1 cells, cytokines responded in distinct patterns upon LPS stimulation in a time- and dose-dependent manner, and the differential regulation of the response to LPS between J774A.1 and THP-1 cells appeared to correlate with the differential regulation of TLR4 at the mRNA level.

Conclusion

In summary, this study indicated that temporal and dose-dependent responses to LPS need to be controlled for and that extrapolation of data on mechanisms may differ between cell lines of different origin.     

IL6 trans-signaling promotes functional recovery of hypofunctional phagocytes through STAT3 activation during peritonitis

Objective

The role of high interleukin 6 (IL6) levels has not been clearly explained in severe sepsis. We show that the augmentation of the IL6 signal by recombinant IL6 receptors (rIL6R) delivery allows the functional recovery of phagocytes in a peritonitis mouse model.

Materials and Methods

Mice were challenged intraperitoneally (i.p.) with live Staphylococcus aureus for effect of IL6R delivery on the 24 h-survival, bacterial clearance and cellular responses. In additional experiments to assess the effect of IL6R delivery on phagocytosis, the model was i.p. inoculated with heat-killed S. aureus with or without rIL6R and the peritoneal lavage fluid and cells were collected at 1 h after the i.p. inoculation of S. aureus.

Results

The IL6R delivery tended to improve 24 h survival and increase bacteria clearance from the septic mice. The rIL6R treatment to heat-killed bacteria challenged mice augmented the uptake of bacteria and phagosome acidification, inducing the phosphorylation of STAT3 in peritoneal cells within 1 h after the IL6R delivery. Furthermore, the rIL6R delivery prevented the extracellular release of neutrophil elastase activity and myeloperoxidase (harmful factors).

Conclusions

These results indicate that augmentation of IL6 signaling appears to be critical for the effective management of hypofunctional neutrophils during severe inflammation, such as sepsis.