Migration Of Polymorphonuclear Leukocytes Research Articles

852 Under Agarose Migration Assay - a Method to Study Graded Chemotactic Activity of Leukocytes in Newborn Infants

Background: Preterm newborn infants are at risk of developing chronic inflammation and tissue injury in their lungs and CNS due to prolonged and exaggerated accumulation of leukocytes.Aim: To study migration of leukocytes from newborn infants with a modified under agarose cell migration assay.Methods: Blood was collected from healthy adults (n=9) and from healthy, term newborn infants (umbilical cord; caesarean section; spinal anaesthesia; n=9). Isolated leukocytes were added to outer wells and chemoattractants to central wells. Gels were incubated for 2 hours whereby only polymorphonuclear (PMN) leukocytes migrated. Dose responses of intermediate (n=4; IL-8) and end-target (n=5; fMLP) chemoattractants were tested on PMN.Results: Similar dose response curves of PMN migration to gradients of IL-8 and fMLP were observed in newborn infants as in adults, but with less PMN migrating with newborns than with adults. The PMN leukocyte migration per distance in newborns was 80% of adults with IL-8, and >90% of adults with fMLP. There was a high linear correlation between the number of PMN and the distance of migration irrespective of chemoattractant studied (IL-8: r=0.85 and r=0.65 respectively for newborns and adults; p< 0.001 for both) (fMLP: r=0.76 and r=0.87; p< 0.001). The pattern of migration differed between fMLP and IL-8.Conclusion: Similar graded chemotaxis of PMN was observed with under agarose cell migration assay in newborn infants as in adults, making this an attractive method for functional clinical studies of leukocytes in newborn infants during different stages of maturity and disease.

Anti-inflammatory and immunomodulatory effects of RDV-8 [C18H22N2O2S (ethyl 1-butyl-6-methyl-2-phenyl-4-thioxo-1,4-dihydropyrimidine-5-carboxylate)] in a rat model of induced pleurisy and in vitro lymphoproliferation

RDV-8 [C(18)H(22)N(2)O(2)S (ethyl 1-butyl-6-methyl-2-phenyl-4-thioxo-1,4-dihydropyrimidine-5-carboxylate)] is derived from the 4-thioxopyrimidine, and presents important clinical effects. The present study explored the RDV-8 effects in the proliferation of human peripheral blood mononuclear cells (PBMCs), as well as in a pleurisy-induced rat model. PBMCs were directly plated in four different RDV-8 concentrations (0.0125, 0.025, 0.05 and 0.1 mg/mL). RDV-8 decreased cell proliferation and monocyte chemotactic protein 1 synthesis. The interleukin 1 levels and the cytotoxic effect were not significantly affected by RDV-8 treatment. In the carrageenan-induced pleurisy model, the RDV-8 (3 mg/kg) treatment induced a significant reduction in the exudate volume, in the polymorphonuclear leukocyte migration and in the pleural exudate NO levels. The results indicate that RDV-8 may have an immunomodulatory effect, as well as anti-inflammatory actions suggesting that it could represent a new strategy in the inflammatory response modulation.

Shiga Toxin 2 and Flagellin from Shiga-ToxigenicEscherichia coliSuperinduce Interleukin-8 through Synergistic Effects on Host Stress-Activated Protein Kinase Activation

Shiga toxins expressed in the intestinal lumen during infection with Shiga-toxigenic Escherichia coli must translocate across the epithelium and enter the systemic circulation to cause systemic (pathological) effects, including hemolytic uremic syndrome. The transepithelial migration of polymorphonuclear leukocytes in response to chemokine expression by intestinal epithelial cells is thought to promote uptake of Stx from the intestinal lumen by compromising the epithelial barrier. In the present study, we investigated the hypothesis that flagellin acts in conjunction with Shiga toxin to augment this chemokine expression. We investigated the relative contributions of nuclear factor kappaB (NF-kappaB) and mitogen-activated protein kinase (MAPK) signaling to transcription and translation of interleukin-8. Using reporter gene constructs, we showed that flagellin-mediated interleukin-8 gene transcription is heavily dependent on both NF-kappaB and extracellular signal-regulated kinase 1 and 2 (ERK-1/2) activation. In contrast, inhibition of p38 has no detectable effect on interleukin-8 gene transcription, even though flagellin-mediated activation of host p38 is critical for maximal interleukin-8 protein expression. Inhibition of MAPK-interacting kinase 1 suggests that p38 signaling affects the posttranscriptional regulation of interleukin-8 protein expression induced by flagellin. Cotreatment with Stx2 and flagellin results in a synergistic upregulation of c-Jun N-terminal protein kinases (JNKs), p38 activation, and a superinduction of interleukin-8 mRNA. This synergism was also evident at the protein level, with increased interleukin-8 protein detectable following cotreatment with flagellin and Stx2. We propose that flagellin, in conjunction with Shiga toxin, synergistically upregulates stress-activated protein kinases, resulting in superinduction of interleukin-8 and, ultimately, absorption of Stx into the systemic circulation.

Pretreatment with lipopolysaccharide modulates innate immunity in corneal fibroblasts challenged with Aspergillus fumigatus

Aspergillus fumigatus triggers inflammatory responses through Toll-like receptors (TLRs), particularly TLR2 and TLR4. In this study, we tested the hypothesis that lipopolysaccharide (LPS), a ligand of TLR4, can induce tolerance of A. fumigatus hyphae in telomerase-immortalized human stroma fibroblasts (THSFs). The THSFs were pretreated with low-dose LPS for various times and then challenged with A. fumigatus hyphae. We observed that pretreatment of THSFs with low-dose LPS resulted in diminished production of cytokines IL-8 and IL-6 and elevated expression of antimicrobial peptides, such as CC chemokine-ligand 20 (CCL20) and thymosin β4 (Tβ4), upon subsequent A. fumigatus challenge. Furthermore, LPS pretreatment also resulted in suppression of polymorphonuclear leukocyte (PMN) migration. Our results suggested that THSFs pretreated with LPS develop a state of A. fumigatus hyphae tolerance. This may induce protective mechanisms during fungal keratitis to prevent an excessive inflammatory response and to control infection of the cornea.

Pathobiology of the neutrophil-intestinal epithelial cell interaction: Role in carcinogenesis

The role of chronic inflammation, acting as an independent factor, on the onset of gastrointestinal carcinogenesis is now well accepted. However, even if there is an increase in the number of elements directly involving polymorphonuclear leukocytes (PMNL), as a major actor in digestive carcinogenesis, the different cellular and molecular events occurring in this process are still not completely understood. The transepithelial migration of PMNL, which is the ultimate step of the afflux of PMNL into the digestive mucosa, is a complex phenomenon involving sequential interaction of molecules expressed both on PMNL and on digestive epithelial cells. Chronic inflammatory areas rich in PMNL [so-called (chronic active inflammation)] and iterative transepithelial migration of PMNL certainly evoke intracellular signals, which lead toward progressive transformation of epithelia. Among these different signals, the mutagenic effect of reactive oxygen species and nitrates, the activation of the nuclear factor-κB pathway, and the modulation of expression of certain microRNA are key actors. Following the initiation of carcinogenesis, PMNL are involved in the progression and invasion of digestive carcinomas, with which they interact. It is noteworthy that different subpopulations of PMNL, which can have some opposite effects on tumor growth, in association with different levels of transforming growth factor-β and with the number of CD8 positive T lymphocytes, could be present during the development of digestive carcinoma. Other factors that involve PMNL, such as massive elastase release, and the production of angiogenic factors, can participate in the progression of neoplastic cells through tissues. PMNL may play a major role in the onset of metastases, since they allow the tumor cells to cross the endothelial barrier and to migrate into the blood stream. Finally, PMNL play a role, alone or in association with other cell parameters, in the initiation, promotion, progression and dissemination of digestive carcinomas. This review focuses on the main currently accepted cellular and molecular mechanisms that involve PMNL as key actors in digestive carcinogenesis.

Prolonged infection by Fonsecaea pedrosoi after antigenic co-stimulation at different sites in experimental murine chromoblastomycosis

In the present study, we examined prolonged infection after antigenic co-stimulation by inoculation of the fungus Fonsecaea pedrosoi at two different sites in three mouse strains (BALB/c, Swiss, and C57BL/6). Using this murine model of infection, we showed that antigen induction of infection at more than one site led to a local suppression of active lesions, which increased the time course of experimental chromoblastomycosis (CBM). Footpad infection with a simultaneous infection of the peritoneum or a mucosal site appeared to cause prolonged infection and frequent fungal disseminations. Using knockout (KO) mice, we observed that antigenic co-stimulation caused progressive illness in CD8-KO animals and an effective immune response in the absence of IL-10. In Xid mice, co-stimulation provoked chronic infection (not prolonged), suggesting that B1 B cells play an important role in the control of fungal infection. The tissue response to infection was similar in all co-stimulated mouse groups, as anatomopathologic sections revealed multifocal lesions (granuloma-like). In general, these mice had acute responses at primary antigenic sites with an intense migration of polymorphonuclear leukocytes (PMNs), whereas the distant infection sites (footpad) showed signs of chronic infection. The migration of PMNs to the secondary site (footpad) increased in the later periods of infection, especially after the disappearance of the primary antigenic focus. PMN migration was associated with lesion-dormancy breakage and fungal elimination. Our findings suggest that the host inflammatory/suppression mechanisms induced by antigenic co-stimulation to systemically fight the same pathogen act coordinately through responses that differ at the sites of infection between acute and chronic integrated healing processes that are more prolonged than an acute infection at a single site. However, the long persistence of fungal cells in the host may be linked to microbial adaptation to a parasitic infection as observed in co-stimulated Xid mice.

Subgingival biofilm formation

The human body contains numerous distinctive ecosystems that provide a unique environment for colonizing microorganisms. The periodontal pocket is one such microniche. This environment is partially sheltered from the physical shear forces in the oral cavity and contains the hard, nonshedding surfaces of the tooth root along with the shedding surfaces of gingival mucosa. The junctional epithelium, which is attached to the tooth root, is poorly differentiated, lacks keratinization and has relatively wide intercellular spaces. Consequently, junctional epithelium is permeable and allows the migration of polymorphonuclear leukocytes into the periodontal pocket. Furthermore, the tissues in the periodontal pocket are bathed in gingival crevicular fluid, a serum exudate with antioxidant properties. The initial bacterial colonizers attach to the available surfaces, as discussed elsewhere in this volume of Periodontology 2000. Later colonizers attach to the antecedent organisms and assemble into polymicrobial communities. The biofilms on the hard surfaces develop into spatially organized structures that can extend several hundred micrometers from the surface. By contrast, the epithelial surfaces, which are continually being sloughed and replenished, tend to be colonized with monolayers of microorganisms. However, several of the more pathogenic species of bacteria are able to invade the gingival cells and tissues where they can remain viable and thus constitute a nidus of infection. Interspecies adherence interactions help to shape the temporal and spatial development of the complex bacterial consortia in the gingival crevice. Bacteria within these communities encounter high cell densities and, in consequence, community living involves adaptation to higher (and unevenly distributed) levels of metabolic by-products, secondary metabolities and other secreted molecules, and to the sporadic availability of nutrients and oxygen. Bacterial inhabitants of biofilms are known to both collaborate (e.g. through nutritional cross-feeding) and compete (e.g. through production of bacteriocins) as they strive to optimize their adaptation to these environmental constraints. Bacteria can also communicate with one another through a variety of sensing and response systems based on either cell-to-cell contact or detection of soluble mediators. The signaling molecules are processed through transcriptional and post-transcriptional networks and they allow bacterial inhabitants of biofilms to coordinate activities at a group or community level. An understanding of the mechanisms of subgingival biofilm formation and development needs, therefore, to accommodate the multiple interspecies interactions that occur in polymicrobial communities.

Streptolysin S inhibits neutrophil recruitment during the early stages of Streptococcus pyogenes infection.

In contrast to infection of superficial tissues, Streptococcus pyogenes infection of deeper tissue can be associated with a significantly diminished inflammatory response, suggesting that this bacterium has the ability to both promote and suppress inflammation. To examine this, we analyzed the behavior of an S. pyogenes mutant deficient in expression of the cytolytic toxin streptolysin S (SLS-) and evaluated events that occur during the first few hours of infection by using several models including injection of zebrafish (adults, larvae, and embryos), a transepithelial polymorphonuclear leukocyte (PMN) migration assay, and two-photon microscopy of mice in vivo. In contrast to wild-type S. pyogenes, the SLS- mutant was associated with the robust recruitment of neutrophils and significantly reduced lethal myositis in adult zebrafish. Similarly, the mutant was attenuated in embryos in its ability to cause lethality. Infection of larva muscle allowed an analysis of inflammation in real time, which revealed that the mutant had recruited PMNs to the infection site. Analysis of transepithelial migration in vitro suggested that SLS inhibited the host cells' production of signals chemotactic for neutrophils, which contrasted with the proinflammatory effect of an unrelated cytolytic toxin, streptolysin O. Using two-photon microscopy of mice in vivo, we showed that the extravasation of neutrophils during infection with SLS- mutant bacteria was significantly accelerated compared to infection with wild-type S. pyogenes. Taken together, these data support a role for SLS in the inhibition of neutrophil recruitment during the early stages of S. pyogenes infection.

CORM-3-derived CO modulates polymorphonuclear leukocyte migration across the vascular endothelium by reducing levels of cell surface-bound elastase

Recently, it has been shown that carbon monoxide (CO)-releasing molecule (CORM)-released CO can suppress inflammation. In this study, we assessed the effects and potential mechanisms of a ruthenium-based water-soluble CO carrier [tricarbonylchloroglycinate-ruthenium(II) (CORM-3)] in the modulation of polymorphonuclear leukocyte (PMN) inflammatory responses in an experimental model of sepsis. Sepsis in mice was induced by cecal ligation and puncture. CORM-3 (3 mg/kg iv) was administered 15 min after the induction of cecal ligation and puncture. PMN accumulation in the lung (myeloperoxidase assay), bronchoalveolar lavage (BAL) fluid, and lung vascular permeability (protein content in BAL fluid) were assessed 6 h later. In in vitro experiments, human PMNs were primed with LPS (10 ng/ml) and subsequently stimulated with formyl-methionyl-leucylphenylalanine (fMLP; 100 nM). PMN production of ROS (L-012/dihydrorhodamine-123 oxidation), degranulation (release of elastase), and PMN rolling, adhesion, and migration to/across human umbilical vein endothelial cells (HUVECs) were assessed in the presence or absence of CORM-3 (1-100 muM). The obtained results indicated that systemically administered CORM-3 attenuates PMN accumulation and vascular permeability in the septic lung. Surprisingly, in in vitro experiments, treatment of PMNs with CORM-3 further augmented LPS/fMLP-induced ROS production and the release of elastase. The latter effects, however, were accompanied by an inability of PMNs to mobilize elastase to the cell surface (plasma membrane), an event required for efficient PMN transendothelial migration. The CORM-3-induced decrease in cell surface levels of elastase was followed by decreased PMN rolling/adhesion to HUVECs and complete prevention of PMN migration across HUVECs. In contrast, treatment of HUVECs with CORM-3 had no effect on PMN transendothelial migration. Taken together, these findings indicate that, in sepsis, CORM3-released CO, while further amplifying ROS production and degranulation of PMNs, concurrently reduces the levels of cell surface-bound elastase, which contributes to suppressed PMN transendothelial migration.

Selective NF-κB inhibition, but not dexamethasone, decreases acute lung injury in a newborn piglet airway inflammation model

Acute respiratory failure in neonates (e.g. ARDS, meconium aspiration pneumonitis, pneumonia) is characterized by an excessive inflammatory response, governing the migration of polymorpho-nuclear leukocytes (PMNLs) into lung tissue and causing consecutive impairment of gas exchange and lung function. Critical to this inflammatory response is the activation of nuclear factor-κB (NF-κB) that is required for transcription of the genes for many pro-inflammatory mediators. We asked whether the inhibition of NF-κB activity using either a selective inhibitor (IKK-NBD peptide) or dexamethasone would be more effective in decreasing NF-κB activity and chemokine expression in pulmonary cells. Changes in lung function were repeatedly assessed for 24 h following induction of acute respiratory failure and therapeutic intervention. We conducted a randomized, controlled, prospective animal study with mechanically ventilated newborn piglets which underwent repeated airway lavage (20 ± 2 [SEM]) to remove surfactant and to induce lung inflammation. Admixed to 100 mg kg −1 surfactant, piglets then received either IKK-NBD peptide (S+IKK), a selective inhibitor of NF-κB activation, its control peptide without intrinsic activity, dexamethasone (S+Dexa), its solvent aqua, or an air bolus only (all groups n = 8). After 24 h of mechanical ventilation, the following differences were measured: PaO 2/FiO 2 (S+IKK 230 ± 9 mmHg vs. S+Dexa 188 ± 14, p < 0.05); ventilation efficiency index (0.18 ± 0.01 [3800/(PIP-PEEP) *f *PaCO 2] vs. 0.14 ± 0.01, p < 0.05); extravascular lung water (24 ± 1 ml kg −1 vs. 29 ± 2, p < 0.05); PMNL in BAL fluid (112 ± 21 cells μl −1 vs. 208 ± 34, p < 0.05), IL-8 (351 ± 117 pg ml −1 vs. 491 ± 144, p = ns) and leukotriene B 4 (23 ± 7 pg ml −1 vs. 71 ± 11, p < 0.01) in BAL fluid. NF-κB activity in the nucleus of pulmonary cells differed by 32 ± 5% vs. 55 ± 3, p < 0.001. Differences between these two intervention groups were more pronounced in the second half of the observation period (hours 12–24). At 24 h of mechanical ventilation, inhibition of NF-κB activity by IKK-NBD peptide admixed to surfactant as a carrier caused improved gas exchange, lung function and reduced pulmonary inflammation, as evidenced by reduction in PMNL migration into lung tissue due to reduced nuclear NF-κB activity. We conclude that IKK-NBD admixture to surfactant in acute neonatal respiratory failure is superior to dexamethasone administration within the first 24 h.

The Heparan Sulfate Proteoglycan Form of Epithelial CD44v3 Serves as a CD11b/CD18 Counter-receptor during Polymorphonuclear Leukocyte Transepithelial Migration

Leukocyte beta2-integrin CD11b/CD18 mediates the firm adhesion and subsequent transepithelial migration of polymorphonuclear leukocytes, but the identity of its counter-receptor(s) on epithelia remains elusive. Here we identified a monoclonal antibody, clone C3H7, which strongly bound to the basolateral membranes of epithelial cells and inhibited both the adhesion of epithelial cells to immobilized CD11b/CD8 and the transepithelial migration of PMNs in a physiologically relevant basolateral-to-apical direction. C3H7 antigen expression in epithelial monolayers was significantly increased by treatment with proinflammatory cytokine interferon-gamma or a combination of interferon-gamma and tumor necrosis factor-alpha. Up-regulation of C3H7 antigen was also observed in the epithelium of inflamed human colon tissues. Microsequencing and Western blotting of the purified antigen showed it to be CD44 variant 3 (CD44v3), a approximately 160-kDa membrane glycoprotein. Further studies demonstrated that this epithelial CD44v3 specifically binds to CD11b/CD18 through its heparan sulfate moieties. In summary, our study demonstrates for the first time that the heparan sulfate proteoglycan form of epithelial CD44v3 plays a critical role in facilitating PMN recruitment during inflammatory episodes via directly binding to CD11b/CD18.

Ultrastructural Alterations In The Epidermis Of Patients With Tinea Pedis

Objective: Tinea pedis is the most common superficial fungal infection of the foot. Although light microscopic characteristics of tinea pedis have already been described and are well known, electron microscopic data is still lacking. In this study, we aimed to examine the ultrastructural changes in the epidermis of patients diagnosed with tinea pedis. Material and Methods: Biopsies were taken from the lesions between the toes of patients with untreated tinea pedis and from healthy volunteers with no fungal infections. The materials obtained were prepared for electron microscopy and examined by transmission electron microscope. Results: The ultrastructural examination revealed the following changes: (1) Disturbances in the form and organization of keratinocytes; (2) Irregular distribution and interlacing of tonofilament bundles in keratinocytes; (3) Disruption of desmosomes and detachment of adjoining keratinocytes; (4) Excessive widening of intercellular spaces between keratinocytes; (5) Dilatation of intercellular spaces between basal cells; (6) Degranulation of melanocytes in the stratum basale; (7) Migration of lymphocytes and polymorphonuclear leukocytes between keratinocytes in the stratum spinosum; (8) Degradation of basal lamina; (9) Pericapillary edema in the apillary dermis. Conclusion: The ultrastructural findings in tinea pedis are described and related to the clinical symptoms and histopathologic features of the disease.

Ca2+ Entry via TRPC Channels Is Necessary for Thrombin-induced NF-κB Activation in Endothelial Cells through AMP-activated Protein Kinase and Protein Kinase Cδ

The transient receptor potential canonical (TRPC) family channels are proposed to be essential for store-operated Ca2+ entry in endothelial cells. Ca2+ signaling is involved in NF-kappaB activation, but the role of store-operated Ca2+ entry is unclear. Here we show that thrombin-induced Ca2+ entry and the resultant AMP-activated protein kinase (AMPK) activation targets the Ca2+-independent protein kinase Cdelta (PKCdelta) to mediate NF-kappaB activation in endothelial cells. We observed that thrombin-induced p65/RelA, AMPK, and PKCdelta activation were markedly reduced by knockdown of the TRPC isoform TRPC1 expressed in human endothelial cells and in endothelial cells obtained from Trpc4 knock-out mice. Inhibition of Ca2+/calmodulin-dependent protein kinase kinase beta downstream of the Ca2+ influx or knockdown of the downstream Ca2+/calmodulin-dependent protein kinase kinase beta target kinase, AMPK, also prevented NF-kappaB activation. Further, we observed that AMPK interacted with PKCdelta and phosphorylated Thr505 in the activation loop of PKCdelta in thrombin-stimulated endothelial cells. Expression of a PKCdelta-T505A mutant suppressed the thrombin-induced but not the TNF-alpha-induced NF-kappaB activation. These findings demonstrate a novel mechanism for TRPC channels to mediate NF-kappaB activation in endothelial cells that involves the convergence of the TRPC-regulated signaling at AMPK and PKCdelta and that may be a target of interference of the inappropriate activation of NF-kappaB associated with thrombosis.

Decrease in adhesion molecules on polymorphonuclear leukocytes of patients with fibromyalgia

Fibromyalgia (FM) is a chronic widespread pain condition in highly stressed humans. Because stress is known to modulate adhesion molecule expression, we determined L: -selectin (CD62L) and beta(2)-integrin (CD11b/CD18) expression on the surface of polymorphonuclear leukocytes in 22 patients with FM. As compared to age and sex-matched healthy controls, FM patients showed a significantly decreased expression of CD62L (p < 0.01) and CD11b/CD18 (p < 0.05) on polymorphonuclear leukocytes. These changes might lower the rate of polymorphonuclear leukocyte migration to sites of inflammation and thereby compromise defense against infections and pain control.

Antiinflammatory effects of etoricoxib alone and combined with NSAIDs in LPS-induced reactive arthritis

Nonsteroidal anti-inflammatory drugs constitute the primary therapeutic approach in reactive arthritis. Here, we compared etoricoxib, a specific COX-2 inhibitor, with other cyclooxygenase inhibitors on articular incapacitation, edema, leukocyte migration, and gastric damage, in a model of LPS-induced reactive arthritis in rats. E. coli Lipopolysaccharide was injected into a carrageenan-primed knee-joint of rats. The effects of etoricoxib, piroxicam, indomethacin, as well the combination of etoricoxib either with piroxicam or indomethacin, were evaluated on articular incapacitation and edema. Afterwards, the synovial leukocyte ontent and the stomach bleeding points were counted. Etoricoxib, piroxicam, and indomethacin dose-dependently inhibited incapacitation and edema. However, only etoricoxib inhibited both mononuclear and polymorphonuclear leukocyte migration. Piroxicam inhibited only mononuclear migration, while indomethacin even increased polymorphonuclear content in inflamed synovia. Associating etoricoxib with either subeffective doses of piroxicam or indomethacin did not enhance the hyponociceptive or the antiedematogenic effect, but prevented the anti-leukocyte migration effect and increased gastric damage. The present results suggest that the selective COX-2 inhibitor etoricoxib could be a better option than non-selective COX inhibitors, since it presented a potent inhibitory effect on the clinical signals and also a potent inhibition on cell migration.

The role of tumour necrosis factor-alpha and IL-1 in polymorphonuclear leucocyte and T lymphocyte recruitment to joint inflammation in adjuvant arthritis.

The mediators involved in leucocyte recruitment to joints during arthritis are not fully defined, but two important proinflammatory cytokines, IL-1 and tumour necrosis factor-alpha (TNF-alpha), are produced in joints in rheumatoid arthritis (RA). We investigated in the rat adjuvant arthritis model whether endogenous IL-1 and TNF-alpha contribute to joint inflammation and polymorphonuclear leucocyte (PMNL) and T lymphocyte infiltration. The migration of 51Cr-labelled rat blood PMNL and 111In-labelled T lymphocytes to the joints of rats with adjuvant arthritis was measured along with plasma protein extravasation, which was quantified using 125I-labelled human albumin. Rats with active arthritis of 5 days' duration received i.p. non-immune serum, polyclonal neutralizing anti-serum to rat TNF-alpha, antiserum to IL-1 alpha and IL-1 beta, or both anti-TNF plus anti-IL-1 for 5 days. Treatment with anti-IL-1 alpha and IL-1 beta did not affect plasma protein extravasation, or PMNL or T lymphocyte accumulation in the joints (i.e. talar joint, hind paws, and tail) despite the fact that this treatment inhibited 80-90% of the PMNL migration into dermal sites injected with IL-1 alpha or IL-1 beta. In contrast, anti-TNF-alpha treatment significantly improved clinical scores, decreased plasma protein extravasation by 60-80%, inhibited PMNL accumulation by 40-50% and decreased T lymphocyte accumulation by 30-50%. Treatment with anti-IL-1, together with anti-TNF-alpha, significantly potentiated the inhibition of T lymphocyte accumulation observed with anti-TNF-alpha alone. These results indicate that endogenous TNF-alpha production may play an important role in the inflammatory changes and leucocyte recruitment in this experimental model of human arthritis, while IL-1 may have a less important role in leucocyte recruitment to these joints.

Polymorphonuclear leukocyte (PMN) migration across vascular endothelial cells (EC) in the absence or presence of IL‐1β differentially regulate expression of EC adhesion molecules ICAM‐1 and E‐selectin

Our findings indicate that PMN migration across activated EC results in down‐regulation of EC nuclear transcription factor, NFκB. However, the subsequent functional consequences are largely unknown.In this study we assessed the effects of PMN transendothelial migration on NFκB‐dependent expression of EC adhesion molecules, ICAM‐1 and E‐selectin during inflammation.To this end human endothelial cells (HUVEC) were grown in Traswell inserts and stimulated with IL‐1β (1ng/ml; 4 hrs). Subsequently, PMN were added and allowed to migrate across HUVEC in response to fMLP (10−8M) chemotactic gradient in the absence (i.e. across washed HUVEC) or presence of IL‐1β. ICAM‐1 and E‐selectin gene expression in EC was assessed by RT‐PCR after 4 or 24 hrs following PMN migration.The obtained results indicate that migration of PMN across IL‐1β treated and subsequently washed HUVEC results in a marked down‐regulation of IL‐1 β‐induced ICAM‐1, and to a lesser degree, E‐selectin gene expression as assessed 4 and 24 hrs following PMN migration. On the contrary, migration of PMN across activated HUVEC in the presence of IL‐1β results in a further increase in both, ICAM‐1 and E‐selectin expression.Taken together these findings indicate that migrating PMN differentially modulate expression of EC adhesion molecules during inflammation and can offer EC with both, anti‐ and pro‐inflammatory stimuli (HSFO‐NA5580/NA6171)

Activated polymorphonuclear cells promote injury and excitability of dorsal root ganglia neurons

Therapies aimed at depleting or blocking the migration of polymorphonuclear leukocytes (PMN or neutrophils) are partially successful in the treatment of neuroinflammatory conditions and in attenuating pain following peripheral nerve injury or subcutaneous inflammation. However, the functional effects of PMN on peripheral sensory neurons such as dorsal root ganglia (DRG) neurons are largely unknown. We hypothesized that PMN are detrimental to neuronal viability in culture and increase neuronal activity and excitability. We demonstrate that isolated peripheral PMN are initially in a relatively resting state but undergo internal oxidative burst and activation by an unknown mechanism within 10 min of co-culture with dissociated DRG cells. Co-culture for 24 h decreases neuronal count at a threshold < 0.4:1 PMN:DRG cell ratio and increases the number of injured and apoptotic neurons. Within 3 min of PMN addition, fluorometric calcium imaging reveals intracellular calcium transients in small size (< 25 μm diam) and large size (> 25 μm diam) neurons, as well as in capsaicin-sensitive neurons. Furthermore, small size isolectin B4-labeled neurons undergo hyperexcitability manifested as decreased current threshold and increased firing frequency. Although co-culture of PMN and DRG cells does not perfectly model neuroinflammatory conditions in vivo, these findings suggest that activated PMN can potentially aggravate neuronal injury and cause functional changes to peripheral sensory neurons. Distinguishing the beneficial from the detrimental effects of PMN on neurons may aid in the development of more effective drug therapies for neurological disorders involving neuroinflammation, including painful neuropathies.

Saccharomyces boulardiiinterferes withShigellapathogenesis by postinvasion signaling events

Saccharomyces boulardii is gaining in popularity as a treatment for a variety of diarrheal diseases as well as inflammatory bowel disease. This study was designed to examine the effect of this yeast on infection by Shigella flexneri, a highly infectious and human host-adapted enteric pathogen. We investigated key interactions between the bacteria and host cells in the presence of the yeast in addition to a number of host responses including proinflammatory events and markers. Although the presence of the yeast during infection did not alter the number of bacteria that was able to attach or invade human colon cancer-derived T-84 cells, it did positively impact the tight junction protein zonula occluden-2 and significantly increase the barrier integrity of model epithelia. The yeast also decreased ERK, JNK, and NF-kappaB activation in response to S. flexneri, events likely responsible for the observed reductions in IL-8 secretion and the transepithelial migration of polymorphonuclear leukocytes across T-84 monolayers. These results, suggesting that the yeast allowed for a dampened inflammatory response, were confirmed in vivo utilizing a highly relevant model of human fetal colonic tissue transplanted into scid mice. Furthermore, a cell-free S. boulardii culture supernatant was also capable of reducing IL-8 secretion by infected T-84 cells. These data suggest that although the use of S. boulardii during infection with S. flexneri may alleviate symptoms associated with the inflammatory response of the host, it would not prevent infection.

Neutrophil Elastase Converts Human Immature Dendritic Cells into Transforming Growth Factor-β1-Secreting Cells and Reduces Allostimulatory Ability

During microbial infection, neutrophils (polymorphonuclear leukocytes; PMNs) activate dendritic cells (DCs). However, early reports illustrated that neutrophil-derived mediators may suppress responses to mitogens. In the present study, we investigated the mechanism used by PMNs to modulate the immunostimulatory ability of DCs. Autologous syngeneic PMNs decreased T-cell proliferation induced by allogeneic DCs. Culture supernatant (CS) derived from PMNs also decreased allostimulation ability of immature DCs and increased the expression of transforming growth factor (TGF)-beta1 on DCs. A TGF-beta1 monoclonal antibody, a CD40 monoclonal antibody, or a serine protease inhibitor reversed the effect of PMN CS on DC allostimulatory ability. Furthermore, elastase reproduced the inhibitory effect of PMN CS on DC allostimulatory ability and the TGF-beta1 production. The role of elastase was confirmed by examining PMN CS from two patients with cyclic neutropenia, a disease due to mutations in the neutrophil elastase gene. These PMN CS samples had reduced elastase activity and were unable to increase DC TGF-beta1 production. Moreover, elastase and PMN CS induced IkappaBalpha degradation in DCs. We conclude that PMNs decrease DC allostimulatory ability via production of elastase leading to a switch of immature DCs into TGF-beta1-secreting cells.