Early Accumulation Of Neutrophils Research Articles

Novel murine model reveals an early role for pertussis toxin in disrupting neonatal immunity to Bordetella pertussis

Abstract The increased susceptibility of neonates to some pathogens has previously been attributed to an underdeveloped immune system. More recent data suggest neonates have effective protection against most pathogens but are particularly susceptible to those that target immune functions specific to neonates. Bordetella pertussis (Bp), the causative agent of “whooping cough”, causes more serious disease in infants attributed to its production of pertussis toxin (PTx). Problematically, the rapid development of adult immunity in mice has confounded our ability to study the interaction of this agent with neonatal immune components, such as virtual memory T cells which are prominent prior to the maturation of the thymus. Here, we examine the rapid change in susceptibility of young mice and define a period from five- to eight-days-old during which mice are much more susceptible to Bp than mice even a few days older. These more narrowly defined “neonatal” mice display significantly increased susceptibility to wild type Bp but very rapidly and effectively respond to and control Bp lacking PTx, more rapidly even than adult mice. The clearance of the PTx mutant correlates with the early accumulation of neutrophils and T cells and suggests a role for PTx in disrupting their accumulation. These results define an early age of extreme susceptibility to Bp, and demonstrate that the neonatal response can be more efficient than the adult response in eliminating bacteria from the lungs, but these neonatal functions are substantially blocked by PTx. This refined definition of “neonatal” mice may be useful in the study of other pathogens that predate neonates, and PTx may prove a particularly valuable tool for probing the poorly understood neonatal immune system. This work was supported by grants AI156293 and AI159347 of the National Institutes of Health to Eric Harvill and AI139449 of the National Institutes of Health to Nancy Manley.

Novel murine model reveals an early role for pertussis toxin in disrupting neonatal immunity to Bordetella pertussis.

The increased susceptibility of neonates to specific pathogens has previously been attributed to an underdeveloped immune system. More recent data suggest neonates have effective protection against most pathogens but are particularly susceptible to those that target immune functions specific to neonates. Bordetella pertussis (Bp), the causative agent of "whooping cough", causes more serious disease in infants attributed to its production of pertussis toxin (PTx), although the neonate-specific immune functions it targets remain unknown. Problematically, the rapid development of adult immunity in mice has confounded our ability to study interactions of the neonatal immune system and its components, such as virtual memory T cells which are prominent prior to the maturation of the thymus. Here, we examine the rapid change in susceptibility of young mice and define a period from five- to eight-days-old during which mice are much more susceptible to Bp than mice even a couple days older. These more narrowly defined "neonatal" mice display significantly increased susceptibility to wild type Bp but very rapidly and effectively respond to and control Bp lacking PTx, more rapidly even than adult mice. Thus, PTx efficiently blocks some very effective form(s) of neonatal protective immunity, potentially providing a tool to better understand the neonatal immune system. The rapid clearance of the PTx mutant correlates with the early accumulation of neutrophils and T cells and suggests a role for PTx in disrupting their accumulation. These results demonstrate a striking age-dependent response to Bp, define an early age of extreme susceptibility to Bp, and demonstrate that the neonatal response can be more efficient than the adult response in eliminating bacteria from the lungs, but these neonatal functions are substantially blocked by PTx. This refined definition of "neonatal" mice may be useful in the study of other pathogens that primarily infect neonates, and PTx may prove a particularly valuable tool for probing the poorly understood neonatal immune system.

Increased neutrophil infiltration as a body-wide effect in pancreatic cancer development.

Increased neutrophil infiltration as a body-wide effect in pancreatic cancer development.

In vitro Monitoring of Extracellular pH in Real-Time.

Early accumulation of neutrophils (PMN) is a hallmark of acute intestinal inflammation. This acute inflammation is either resolved or progresses to chronic inflammation. Without efficient PMN clearance at sites of infiltration, PMN can accumulate and contribute to chronic inflammatory conditions, including the intestinal diseases ulcerative colitis (UC) and Crohn's Disease (CD). The pH in the distal colon in individuals with active UC can range between a pH of 5 and 6, whereas healthy individuals maintain colonic pH in the range of 6.8-7.4. Extracellular pH has been shown to influence both intestinal epithelial cells and the infiltrating immune cells. More specifically, extracellular acidosis significantly impacts PMN. At pH below 6.5, there are increases in the production of H2O2, inhibition of apoptosis, and increases in the functional lifespan of PMN. Given the significant presence of PMN and extracellular acidification at sites of inflammation, we developed a novel model that allows for the monitoring of extracellular pH during PMN transepithelial migration in real time. Here, we describe this model and how it can be utilized to measure both the apical and basal pH during PMN trafficking. This model can be utilized to monitor extracellular pH under a wide range of conditions; including, hypoxia, PMN transepithelial migration, and for extended periods of time.

Interleukin-38 ameliorates poly(I:C) induced lung inflammation: therapeutic implications in respiratory viral infections

Interleukin-38 has recently been shown to have anti-inflammatory properties in lung inflammatory diseases. However, the effects of IL-38 in viral pneumonia remains unknown. In the present study, we demonstrate that circulating IL-38 concentrations together with IL-36α increased significantly in influenza and COVID-19 patients, and the level of IL-38 and IL-36α correlated negatively and positively with disease severity and inflammation, respectively. In the co-cultured human respiratory epithelial cells with macrophages to mimic lung microenvironment in vitro, IL-38 was able to alleviate inflammatory responses by inhibiting poly(I:C)-induced overproduction of pro-inflammatory cytokines and chemokines through intracellular STAT1, STAT3, p38 MAPK, ERK1/2, MEK, and NF-κB signaling pathways. Intriguingly, transcriptomic profiling revealed that IL-38 targeted genes were associated with the host innate immune response to virus. We also found that IL-38 counteracts the biological processes induced by IL-36α in the co-culture. Furthermore, the administration of recombinant IL-38 could mitigate poly I:C-induced lung injury, with reduced early accumulation of neutrophils and macrophages in bronchoalveolar lavage fluid, activation of lymphocytes, production of pro-inflammatory cytokines and chemokines and permeability of the alveolar-epithelial barrier. Taken together, our study indicates that IL-38 plays a crucial role in protection from exaggerated pulmonary inflammation during poly(I:C)-induced pneumonia, thereby providing the basis of a novel therapeutic target for respiratory viral infections.

Adaptation to inflammatory acidity through neutrophil-derived adenosine regulation of SLC26A3.

Acute intestinal inflammation includes the early accumulation of neutrophils (PMN). Based on recent evidence that PMN infiltration “imprints” changes in the local tissue environment through local oxygen depletion and the release of adenine nucleotides, we hypothesized that the interaction between transmigrating PMN and intestinal epithelial cells (IECs) results in inflammatory acidification of the tissue. Using newly developed tools, we revealed that active PMN transepithelial migration (TEM) significantly acidifies the local microenvironment, a decrease of nearly 2 pH units. Using unbiased approaches, we sought to define acid-adaptive pathways elicited by PMN TEM. Given the significant amount of adenosine (Ado) generated during PMN TEM, we profiled the influence of Ado on IECs gene expression by microarray and identified the induction of SLC26A3, the major apical Cl−/HCO3− exchanger in IECs. Utilizing loss- and gain-of-function approaches, as well as murine and human colonoids, we demonstrate that Ado-induced SLC26A3 promotes an adaptive IECs phenotype that buffers local pH during active inflammation. Extending these studies, chronic murine colitis models were used to demonstrate that SLC26A3 expression rebounds during chronic DSS-induced inflammation. In conclusion, Ado signaling during PMN TEM induces an adaptive tissue response to inflammatory acidification through the induction of SLC26A3 expression, thereby promoting pH homeostasis.

Trehalose-6-phosphate synthase-deletion in Cryptococcus neoformans elicits rapid innate fungal clearance from the lungs with early neutrophil accumulation

Abstract Clearance of the invasive fungus Cryptococcus neoformans (Cn) infection requires T-cell immune responses and individuals with impaired adaptive immunity are highly vulnerable to Cn infection. The innate immune response thus may be the only functional arm against the expansion of pathogens in patients with severe T cell deficiencies. Accordingly, targeting pathways responsible for Cn-evasion of early innate defenses could rapidly resolve Cn-disease in immunocompromised patients. Trehalose-6-phosphate synthase (TPS1), a fungal enzyme for trehalose sugar synthesis absent in mammal, was recently identified as a key virulence gene. Using a mouse model of pulmonary Cn infection, we found that TPS1-deficient Cn (tps1Δ) elicits rapid clearance by the host. Within 72 hours, tps1Δ was either completely cleared or reduced by 5 orders of magnitude, while WT-Cn expanded in the lungs of infected mice. Mechanistically, we found that rapid tps1Δ clearance was associated with early neutrophils recruitment and induction of neutrophils - recruiting CXCL1 and CXCL2. Notably, this occurred without improved Cn-killing by macrophages. Tps1Δ stains were rapidly cleared from CD4+ T cell depleted mice that receive anti-CD4 antibody treatment, suggesting that Tps1Δ clearance is independent of host adaptive immunity. Furthermore, our in vitro killing assay showed that tps1Δ was more vulnerable to neutrophils killing compared to WT strain. Together, our data revealed that cnTPS1 contributes to early innate immune evasion by inhibiting neutrophil recruitment and killing. We identify TPS1-interception as an important therapeutic strategy to both compromise the microbe fitness and to enhance efficiency of the innate defenses in C.n elimination.

Investigation of inflammatory and allergic responses to common mold species: Results from in vitro experiments, from a mouse model of asthma, and from a group of asthmatic patients

Most studies on molds focus on Alternaria alternata and Aspergillus fumigatus. Here, we report on inflammatory and allergenic properties of more typical indoor species Aspergillus versicolor, P.chrysogenum, C.cladosporioïdes, and C.sphaerospermum that were compared to A.alternata and A.fumigatus. In a mouse model, after intranasal instillation, A.alternaria, A.versicolor, and C.sphaerospermum induced the early recruitment of neutrophils and the strong expression of inflammatory markers in the bronchoalveolar lavages fluids. A.fumigatus also induced the early accumulation of neutrophils but with lower levels of inflammatory markers. Chronic treatment induced variable response according to species: P.chrysogenum and A.fumigatus appeared strong pro-allergenic inducers compared to A.alternata and C.sphaerospermum while A.versicolor and C.cladosporioides induced a mixed pro-allergenic/pro-inflammatory response. In mold-sensitized asthmatics, mold-specific Immunoglobulin E (IgE) were detected with an in-house dot-blot assay. A.fumigatus and A.alternata were the most frequent sensitizers. Altogether, P.chrysogenum, P.brevicompactum, C.sphaerospermum, and C.cladosporïoides were the "major sensitizer" (defined as the strongest response against a single mold species) for almost 30% of the asthmatics. These results show that, not only A.alternata and A.fumigatus, but also indoor species have strong inflammatory and allergic properties and a harmful potency.

IL-36 receptor deletion attenuates lung injury and decreases mortality in murine influenza pneumonia.

Influenza virus causes a respiratory disease in human that can progress to lung injury with fatal outcome. The interleukin (IL)-36 cytokines are newly described IL-1 family cytokines that promote inflammatory responses via binding to the IL-36 receptor (IL-36R). The mechanism of expression and the role of IL-36 cytokines is poorly understood. Here, we investigated the role of IL-36 cytokines in modulating the innate inflammatory response during influenza virus-induced pneumonia in mice. The intranasal administration of influenza virus upregulated IL-36α mRNA and protein production in the lungs. In vitro, influenza virus-mediated IL-36α but not IL-36γ is induced and secreted from alveolar epithelial cells (AECs) through both a caspase-1 and caspase-3/7 dependent pathway. IL-36α was detected in microparticles shed from AECs and promoted the production of pro-inflammatory cytokines and chemokines in respiratory cells. IL-36R deficient mice were protected from influenza virus-induced lung injury and mortality. Decreased mortality was associated with significantly reduced early accumulation of neutrophils and monocytes/macrophages, activation of lymphocytes, production of pro-inflammatory cytokines and chemokines and permeability of the alveolar-epithelial barrier in despite impaired viral clearance. Taken together, these data indicate that IL-36 ligands exacerbate lung injury during influenza virus infection.

IL-1α signaling is critical for leukocyte recruitment after pulmonary Aspergillus fumigatus challenge.

Aspergillus fumigatus is a mold that causes severe pulmonary infections. Our knowledge of how A. fumigatus growth is controlled in the respiratory tract is developing, but still limited. Alveolar macrophages, lung resident macrophages, and airway epithelial cells constitute the first lines of defense against inhaled A. fumigatus conidia. Subsequently, neutrophils and inflammatory CCR2+ monocytes are recruited to the respiratory tract to prevent fungal growth. However, the mechanism of neutrophil and macrophage recruitment to the respiratory tract after A. fumigatus exposure remains an area of ongoing investigation. Here we show that A. fumigatus pulmonary challenge induces expression of the inflammasome-dependent cytokines IL-1β and IL-18 within the first 12 hours, while IL-1α expression continually increases over at least the first 48 hours. Strikingly, Il1r1-deficient mice are highly susceptible to pulmonary A. fumigatus challenge exemplified by robust fungal proliferation in the lung parenchyma. Enhanced susceptibility of Il1r1-deficient mice correlated with defects in leukocyte recruitment and anti-fungal activity. Importantly, IL-1α rather than IL-1β was crucial for optimal leukocyte recruitment. IL-1α signaling enhanced the production of CXCL1. Moreover, CCR2+ monocytes are required for optimal early IL-1α and CXCL1 expression in the lungs, as selective depletion of these cells resulted in their diminished expression, which in turn regulated the early accumulation of neutrophils in the lung after A. fumigatus challenge. Enhancement of pulmonary neutrophil recruitment and anti-fungal activity by CXCL1 treatment could limit fungal growth in the absence of IL-1α signaling. In contrast to the role of IL-1α in neutrophil recruitment, the inflammasome and IL-1β were only essential for optimal activation of anti-fungal activity of macrophages. As such, Pycard-deficient mice are mildly susceptible to A. fumigatus infection. Taken together, our data reveal central, non-redundant roles for IL-1α and IL-1β in controlling A. fumigatus infection in the murine lung.

CCR1-Mediated Formation of Liver Microenvironment for Cancer Metastasis

Accumulating evidence suggests that bone marrow-derived myeloid cells constitute the major components of cancer microenvironment and play significant roles in the progression of cancer. We have previously shown that myeloid cells expressing CCR1, a chemokine receptor, are recruited to the microenvironment of disseminated colon cancer cells in the liver (PNAS 2010). These CCR1+ cells promote metastatic colonization of cancer cells in the liver through producing metalloproteinases MMP9 and MMP2. However, the cell lineage and precise characteristics of these myeloid cells were largely unknown. The aim of this study is to characterize the cell types among these myeloid cells expressing CCR1 and/or MMPs.In order to characterize the CCR1-expressing myeloid cells accumulating at liver metastatic foci, we have established bacterial artificial chromosome (BAC)-based transgenic mouse lines in which membrane-targeted Venus fluorescent protein (mVenus) was expressed under the control of Ccr1cis-regulatory elements. A flow cytometric analysis of adult transgenic mouse bone marrow cells showed that expression of the reporter mVenus fluorescence was restricted to the CD11b+ Gr-1+ cells and the level of endogenous Ccr1 mRNA correlated well with that of mVenus fluorescence, suggesting that expression of the reporter precisely reflected that of the endogenous CCR1.Inoculation of CCL9-expressing mouse colon cancer cell line CMT93 into the spleen of syngeneic hosts causes efficient metastatic colonization of the cancer cells in the liver. Employing this model, we monitored the behavior of the Ccr1-mVenus+ myeloid cells in the cancer metastasis microenvironment, and found that CD45+ CD11b+Ccr1-mVenus+ cells purified from liver metastatic foci were mostly neutrophils. In contrast, within CD45+ CD11b+Ccr1-mVenus– fraction, we identified eosinophils, monocytes and fibrocytes. Notably, the major cell types in the metastatic foci were monocytes and fibrocytes, with the latter producing abundant collagen.Chronologically, accumulation of neutrophils and monocytes preceded that of eosinophils and fibrocytes. In Ccr1−/− mice, early accumulation of Ccr1-mVenus+ Gr-1+ cells was significantly compromised and later recruitment of fibrocytes to the metastatic foci was reduced. Antibody-mediated depletion of neutrophils caused a significant reduction in the fibrocyte number at the metastatic foci. These results suggest that CCR1 plays critical roles in early accumulation of neutrophils and subsequent recruitment of fibrocytes.After having identified four types of myeloid cells in the metastatic foci, we determined the expression levels of CCR1, MMP9 and MMP2 mRNAs in each purified population, and found that CCR1 and MMP9 are expressed preferentially by neutrophils, whereas MMP2 is expressed exclusively by monocytes/fibrocytes. Considering the chronological difference in the appearance of myeloid cells in liver metastatic foci, these results suggest that production of MMP9 and MMP2 in succession by different myeloid cells is necessary for disseminated colon cancer cells to colonize in the liver.In summary, these results suggests that CCR1 is required for recruitment of neutrophils in the early phase of colon cancer dissemination and the subsequent accumulation of fibrocytes, and that MMP9 and MMP2 are produced by neutrophils and fibrocytes, respectively, in a successive manner during the formation of liver metastasis. Further investigation of these cancer-driven host reactions in human colon cancer dissemination may lead to effective preventive measures against cancer metastasis. DisclosuresNo relevant conflicts of interest to declare.

Deficiency of Melanoma Differentiation–associated Protein 5 Results in Exacerbated Chronic Postviral Lung Inflammation

Respiratory viral infections can result in the establishment of chronic lung diseases. Understanding the early innate immune mechanisms that participate in the development of chronic postviral lung disease may reveal new targets for therapeutic intervention. The intracellular viral sensor protein melanoma differentiation-associated protein 5 (MDA5) sustains the acute immune response to Sendai virus, a mouse pathogen that causes chronic lung inflammation, but its role in the development of postviral chronic lung disease is unknown. To establish the role of MDA5 in the development of chronic lung disease. MDA5-deficient or control mice were infected with Sendai virus. The acute inflammatory response was evaluated by profiling chemokine and cytokine expression and by characterizing the composition of the cellular infiltrate. The impact of MDA5 on chronic lung pathology and function was evaluated through histological studies, degree of oxygen saturation, and responsiveness to carbachol. MDA5 deficiency resulted in normal virus replication and in a distinct profile of chemokines and cytokines that associated with acute lung neutropenia and enhanced accumulation of alternatively activated macrophages. Diminished expression of neutrophil-recruiting chemokines was also observed in cells infected with influenza virus, suggesting a key role of MDA5 in driving the early accumulation of neutrophils at the infection site. The biased acute inflammatory response of MDA5-deficient mice led to an enhanced chronic lung inflammation, epithelial cell hyperplasia, airway hyperreactivity, and diminished blood oxygen saturation. MDA5 modulates the development of chronic lung inflammation by regulating the early inflammatory response in the lung.

Interactions between Neutrophils, Th17 Cells, and Chemokines during the Initiation of Experimental Model of Multiple Sclerosis

Experimental autoimmune encephalomyelitis (EAE) is an animal model of multiple sclerosis (MS) in which activated T cell and neutrophil interactions lead to neuroinflammation. In this study the expression of CCR6, CXCR2, and CXCR6 in Th17 cells and neutrophils migrating to the brain during EAE was measured, alongside an evaluation of the production of IL-17, IL-23, CCL-20, and CXCL16 in the brain. Next, inflammatory cell subpopulations accumulating in the brain after intracerebral injections of IL-17 or CXCL1, as well as during modulation of EAE with anti-IL-23R or anti-CXCR2 antibodies, were analyzed. Th17 cells upregulate CXCR2 during the preclinical phase of EAE and a significant migration of these cells to the brain was observed. Neutrophils upregulated CCR6, CXCR2, and CXCR6 during EAE, accumulating in the brain both prior to and during acute EAE attacks. Production of IL-17, IL-23, CCL20, and CXCL16 in the CNS was increased during both preclinical and acute EAE. Intracerebral delivery of CXCL1 stimulated the early accumulation of neutrophils in normal and preclinical EAE brains but reduced the migration of Th17 cells to the brain during the preclinical stage of EAE. Modulation of EAE by anti-IL-23R antibodies ameliorated EAE by decreasing the intracerebral accumulation of Th17 cells.

Transmigrating Neutrophils Shape the Mucosal Microenvironment through Localized Oxygen Depletion to Influence Resolution of Inflammation

Acute intestinal inflammation involves early accumulation of neutrophils (PMNs) followed by either resolution or progression to chronic inflammation. Based on recent evidence that mucosal metabolism influences disease outcomes, we hypothesized that transmigrating PMNs influence the transcriptional profile of the surrounding mucosa. Microarray studies revealed a cohort of hypoxia-responsive genes regulated by PMN-epithelial crosstalk. Transmigrating PMNs rapidly depleted microenvironmental O2 sufficiently to stabilize intestinal epithelial cell hypoxia-inducible factor (HIF). By utilizing HIF reporter mice in an acute colitis model, we investigated the relative contribution of PMNs and the respiratory burst to "inflammatory hypoxia" invivo. CGD mice, lacking a respiratory burst, developed accentuated colitis compared to control, with exaggerated PMN infiltration and diminished inflammatory hypoxia. Finally, pharmacological HIF stabilization within the mucosa protected CGD mice from severe colitis. In conclusion, transcriptional imprinting by infiltrating neutrophils modulates the host response to inflammation, via localized O2 depletion, resulting in microenvironmental hypoxia and effective inflammatory resolution.

Monophosphoryl lipid A induced innate immune responses via TLR4 to enhance clearance of nontypeableHaemophilus influenzaeandMoraxella catarrhalisfrom the nasopharynx in mice

Acute otitis media (AOM) is one of the most common infectious diseases in children. Nontypeable Haemophilus influenzae (NTHi) and Moraxella catarrhalis, Gram-negative bacteria, are considered major pathogens of AOM and respiratory tract infections. In this study, we used monophosphoryl lipid A (MPL) as a Toll-like receptor (TLR4) agonist to induce innate immune responses before challenge with NTHi and M.catarrhalis to enhance bacterial clearance from the nasopharynx. Mice were intranasally administered 40, 10, or 1μg of MPL and challenged with NTHi and M.catarrhalis 12 and 24h later. At 6 and 12h after the bacterial challenge, the mice were killed and nasal washes were collected. The numbers of NTHi, M.catarrhalis, and inflammatory cells were quantitated. Inoculation of MPL produced a significant reduction in the number of bacteria recovered from the nasopharynx at 6 and/or 12h after the bacterial challenge, when compared with control mice. The effect was dose dependent. MPL inoculation also induced the early accumulation of neutrophils in the nasopharynx after exposure to bacteria. MPL is effective for eliciting clearance of both NTHi and M.catarrhalis from the nasopharynx. These results indicate the possibility of a new strategy against Gram-negative bacterial infection that involves the stimulation of the innate immune system by TLR4 agonists such as MPL.

An essential role for NF-kappa B in neutrophil-dependent ischemia/reperfusion injury following pancreaticoduodenal transplantation in rats

Objective To investigate the effects of nuclear factor-κB (NF-κB) on reperfusion injury following pancreaticoduodenal transplantation in rots. Methods The (homologous) male Wistar rat model of heterotopic total pancreaticoduodenal transplantation was used. The proline dithiocarbamates (PmDTC) -treated rats received the intravenous injection of ProDTC ( 15 mg/kg) 5 min before and after operation. The content of NF-κBp65 in pancreas nuclear extracts (Westem blot), the expression of TNF-a, MIP-2 and ICAM-1 transcripts (RT-PCR), the concentrations of serum TNF-α and MIP-2 (ELISA), blood sugar (BG) and serum concentrations of amylase and lipase,and pancreas myeloperoxidase (MPO) were analyzed. Results The content of NF-κBp65, the transcription of TNF-α, MIP-2 and ICAM-1, and the release of serum TNF-α and MIP-2 were significantly increased after transplantation both in the untreated and ProDTC-treated rats,while the levels in the ProDTC-treated group were obviously higher than in the untreated group. Administration of ProDTC resulted in a statistically significant decrease in blood sugar [ (9.1±2.8) mmol/L] and serum concentration of lipase [ (192±26) U/L] against the levels in the untreated rats [(13.0±3.1) mmol/L,(297±31) U/L,P<0.05]. Conclusion NF-κB inhibitor ProDTC can decrease the amount of NF-κB, and inhibit the elevation of TNF-α, MIP-2 and ICAM-1 and early neutrophil accumulation in the pancreas. NF-κB inhibitor ProDTC has protective effects on the pancreatic ischemia/reperfusion injury after pancreaticoduodenal transplantation. Key words: Pancreas; Transplantation; Reperfusion injury; NF-κB

Protective Role for CD1d-Reactive Invariant Natural Killer T Cells in Cauterization-Induced Corneal Inflammation

Corneal inflammation can be induced by various stimuli, such as chemical burns, trauma, and acute bacterial infection, and directly impairs visual acuity. Natural killer T (NKT) cells belong to a specialized population of leukocytes that coexpress the T-cell receptor and NK markers. This study examined the role of CD1d-reactive invariant NKT cells in cauterization-induced acute corneal inflammation. The corneas of CD1d-knockout (KO) mice and Jalpha18-KO mice (both of which are NKT cell deficient) and control mice were cauterized with silver nitrate. Corneal edema and opacity were examined, and the phenotypes of the corneal-infiltrating cells were analyzed histologically at 24 hours and by flow cytometry at 96 hours. Reverse transcription-polymerase chain reaction (RT-PCR) was used to determine the expression of vascular endothelial growth factor (VEGF), interferon (IFN)gamma, and tumor necrosis factor (TNF)alpha in the cauterized corneas. The CD1d-KO and Jalpha18-KO mice had significantly greater levels of corneal edema and opacity than did the control mice. Although the number of infiltrating cells was not significantly different at 96 hours, both groups of NKT cell-deficient mice demonstrated increased early neutrophil accumulation at 24 hours and early expression of VEGF, IFNgamma, and TNFalpha. There was no difference in the level of VEGF-induced corneal neovascularization. NKT cells appear to regulate the early accumulation of neutrophils, protect the cornea from excessive inflammation, and maintain corneal clarity. However, in this study, they did not affect the corneal revascularization process induced by VEGF.

Role of interleukin-6 in a glucan-induced model of granulomatous vasculitis

The role of interleukin-6 (IL-6) in granulomatous vasculitis is not well understood. To investigate its involvement in this type of vasculitis a model of glucan-induced pulmonary vasculitis employed interleukin-6 deficient (IL-6 −/−) mice. Briefly, IL-6 −/− mice and C57B/J6 wild type (IL-6 +/+) mice were injected intravenously with a suspension of glucan isolated from the cell wall of bakers yeast which results in a granulomatous vasculitis primarily in the pulmonary vasculature. Histological examination demonstrated no significant difference in the number of infiltrating leukocytes between the IL-6 +/+ and IL-6 −/− glucan-injured mice. Similar numbers of granulomas were noted in both the IL-6 +/+ and IL-6 −/− injured animals, while no granulomas were seen in saline injected control mice. Cells recovered from the bronchoalveolar lavage (BAL) fluid were differentially stained and counted. While there was a significant increase in infiltrating leukocytes recovered from the BAL following glucan-induced injury, there was no significant difference between the IL-6 +/+ and IL-6 −/− mice. In addition, no difference was demonstrated in total protein content in the BAL fluid between IL-6 +/+ and IL-6 −/− mice. However, myeloperoxidase (MPO) activity in the lungs of the IL-6 −/− mice was less than in their IL-6 +/+ counterparts suggesting that these animals have a partial defect in their ability to recruit neutrophils in this model. Studies done to look for levels of other cytokines/chemokines in these animals to compensate for the loss of IL-6 revealed that only IL-10 in the sera ( p < 0.016) and BAL fluid ( p < 0.05) of IL-6 −/− mice was significantly higher then their IL-6 +/+-injured counterparts. These studies suggest that IL-6, while possibly involved in early neutrophil accumulation in this model does not appear critical to the development of the TH-2 mediated granulomatous vasculitis.

TRAF1 regulates recruitment of lymphocytes and, to a lesser extent, neutrophils, myeloid dendritic cells and monocytes to the lung airways following lipopolysaccharide inhalation

Inhaled lipopolysaccharide (LPS) induces an inflammatory response that may contribute to the pathogenesis of asthma and other airway diseases. Here we investigate the role of tumour necrosis factor (TNF) receptor-associated factor 1 (TRAF1) in leucocyte recruitment using a model of LPS-induced lung inflammation in mice. TRAF1(-/-) mice are completely deficient in the recruitment of lymphocytes to the lower respiratory tract after inhalation of LPS. Although TRAF1(-/-) mice display normal early accumulation of neutrophils, dendritic cells and monocytes in the alveolar airspace, they have a significantly reduced recruitment of these cells by 24 hr after inhalation of LPS when compared to wild-type (WT) mice. Despite normal expression of the pro-inflammatory cytokines TNF, interleukin-1 (IL-1) and IL-6 after LPS treatment, TRAF1(-/-) mice displayed decreased expression of intercellular adhesion molecule 1, vascular cell adhesion molecule 1, CCL17 and CCL20 in the lungs, when compared to LPS-treated WT mice. These results suggest that TRAF1 facilitates LPS-induced leucocyte recruitment into the lung airways by augmenting the expression of chemokines and adhesion molecules. Mice lacking TNF receptor 1 (TNFR1) but not TNFR2 show a phenotype similar to the TRAF1(-/-) mice, suggesting that TRAF1 may act downstream of TNFR1. Significantly, we use bone marrow chimeras to demonstrate that expression of TRAF1 by cells resident in the lungs, but not by circulating leucocytes, is necessary for efficient LPS-induced recruitment of leucocytes to the lung airways.

The course of allergen-induced leukocyte infiltration in human and experimental asthma

Although the timing of allergen-induced bronchoconstriction is well defined, there is little information about the kinetics of allergen-induced leukocyte infiltration in asthma and its comparability between human and animal models of asthma. To investigate systematically allergen-induced leukocyte infiltration into the airway lumen in human and experimental asthma by using bronchoalveolar lavage. Patients with allergic asthma were lavaged at different time points as long as 1 week after segmental allergen challenge. Allergen-sensitized mice were lavaged as long as 3 weeks after allergen challenge. Differential cell counts, lymphocyte subsets, and cytokines were assessed in bronchoalveolar lavage fluid. In both models, neutrophil infiltration was a relatively early event (maximum: 18 hours after challenge). In contrast, eosinophil infiltration peaked 42 hours (human model) to 4 days (mouse model) after allergen challenge, paralleled by an IL-5 peak in this period. There were elevated macrophage counts over a period of several days after allergen challenge in both models. Lymphocytes (predominantly CD4+ T cells) peaked 18 hours after challenge in the human model, but not until 2 weeks after challenge in the murine model. Early neutrophil accumulation (within hours after challenge) and delayed eosinophil accumulation (within days after challenge) in the airway lumen are common features of allergen-induced airway inflammation, whereas lymphocyte kinetics are dependent on the asthma model. Similarities in the infiltration kinetics of granulocytes after allergen challenge suggest a common role for these cells in asthma, whereas the presumed orchestration of allergic inflammation by lymphocytes appears to differ between the models.