Timothy Polak

CCR2 and CCR6, but not endothelial selectins, mediate the accumulation of immature dendritic cells within the lungs of mice in response to particulate antigen

Dendritic cells (DC) migrate from sites of inflammation to lymph nodes to initiate primary immune responses, but the molecular mechanisms by which DC are replenished in the lungs during ongoing pulmonary inflammation are unknown. To address this question, we analyzed the secondary pulmonary immune response of Ag-primed mice to intratracheal challenge with the particulate T cell-dependent Ag sheep erythrocytes (SRBC). We studied wild-type C57BL/6 mice and syngeneic gene-targeted mice lacking either both endothelial selectins (CD62E and CD62P), or the chemokine receptors CCR2 or CCR6. DC, defined as non-autofluorescent, MHC class II+CD11cmod cells, were detected in blood, enzyme-digested minced lung, and bronchoalveolar lavage fluid using flow cytometry and immunohistology. Compared with control mice, Ag challenge increased the frequency and absolute numbers of DC, peaking at day 1 in peripheral blood (6.5-fold increase in frequency), day 3 in lung mince (20-fold increase in total DC), and day 4 in bronchoalveolar lavage fluid (55-fold increase in total DC). Most lung DC expressed CD11c, CD11b, and low levels of MHC class II, CD40, CD80, and CD86, consistent with an immature myeloid phenotype. DC accumulation depended in part upon CCR2 and CCR6, but not endothelial selectins. Thus, during lung inflammation, immature myeloid DC from the bloodstream replace emigrating immature DC and transiently increase total intrapulmonary APC numbers. Early DC recruitment depends in part on CCR2 to traverse vascular endothelium, plus CCR6 to traverse alveolar epithelium. The recruitment of circulating immature DC represents a potential therapeutic step at which to modulate immunological lung diseases.

CCR2 Mediates Conventional Dendritic Cell Recruitment and the Formation of Bronchovascular Mononuclear Cell Infiltrates in the Lungs of Mice Infected with Cryptococcus neoformans

Pulmonary clearance of the encapsulated yeast Cryptococcus neoformans requires the development of T1-type immunity. CCR2-deficient mice infected with C. neoformans develop a non-protective T2 immune response and persistent infection. The mechanisms responsible for this aberrant response are unknown. The objective of this study was to define the number, phenotype, and microanatomic location of dendritic cells (DC) residing within the lung of CCR2+/+ or CCR2−/− mice throughout a time course following infection with C. neoformans. Results demonstrate the CCR2-mediated recruitment of conventional DC expressing modest amounts of costimulatory molecules. DC recruitment was preceded by the up-regulation in the lung of the CCR2 ligands CCL2 and CCL7. Colocalization of numerous DC and CD4+ T cells within bronchovascular infiltrates coincided with increased expression of IL-12 and IFN-γ. By contrast, in the absence of CCR2, DC recruitment was markedly impaired, bronchovascular infiltrates were diminished, and mice developed features of T2 responses, including bronchovascular collagen deposition and IL-4 production. Our results demonstrate that CCR2 is required for the recruitment of large numbers of conventional DC to bronchovascular infiltrates in mice mounting a T1 immune response against a fungal pathogen. These findings shed new insight into the mechanism(s) by which DC recruitment alters T cell polarization in response to an infectious challenge within the lung.

Cytotoxic Potential of Lung CD8+ T Cells Increases with Chronic Obstructive Pulmonary Disease Severity and with In Vitro Stimulation by IL-18 or IL-15

Lung CD8+ T cells might contribute to progression of chronic obstructive pulmonary disease (COPD) indirectly via IFN-γ production or directly via cytolysis, but evidence for either mechanism is largely circumstantial. To gain insights into these potential mechanisms, we analyzed clinically indicated lung resections from three human cohorts, correlating findings with spirometrically defined disease severity. Expression by lung CD8+ T cells of IL-18R and CD69 correlated with severity, as did mRNA transcripts for perforin and granzyme B, but not Fas ligand. These correlations persisted after correction for age, smoking history, presence of lung cancer, recent respiratory infection, or inhaled corticosteroid use. Analysis of transcripts for killer cell lectin-like receptor G1, IL-7R, and CD57 implied that lung CD8+ T cells in COPD do not belong to the terminally differentiated effector populations associated with chronic infections or extreme age. In vitro stimulation of lung CD8+ T cells with IL-18 plus IL-12 markedly increased production of IFN-γ and TNF-α, whereas IL-15 stimulation induced increased intracellular perforin expression. Both IL-15 and IL-18 protein expression could be measured in whole lung tissue homogenates, but neither correlated in concentration with spirometric severity. Although lung CD8+ T cell expression of mRNA for both T-box transcription factor expressed in T cells and GATA-binding protein 3 (but not retinoic acid receptor-related orphan receptor γ or α) increased with spirometric severity, stimulation of lung CD8+ T cells via CD3ε-induced secretion of IFN-γ, TNF-α, and GM-CSF, but not IL-5, IL-13, and IL-17A. These findings suggest that the production of proinflammatory cytokines and cytotoxic molecules by lung-resident CD8+ T cells contributes to COPD pathogenesis.

Specific Engagement of TLR4 or TLR3 Does Not Lead to IFN-β-Mediated Innate Signal Amplification and STAT1 Phosphorylation in Resident Murine Alveolar Macrophages

The innate immune response must be mobilized promptly yet judiciously via TLRs to protect the lungs against pathogens. Stimulation of murine peritoneal macrophage (PMφ) TLR4 or TLR3 by pathogen-associated molecular patterns (PAMPs) typically induces type I IFN-β, leading to autocrine activation of the transcription factor STAT1. Because it is unknown whether STAT1 plays a similar role in the lungs, we studied the response of resident alveolar macrophages (AMφ) or control PMφ from normal C57BL/6 mice to stimulation by PAMPs derived from viruses (polyriboinosinic:polyribocytidylic acid, specific for TLR3) or bacteria (Pam3Cys, specific for TLR2, and repurified LPS, specific for TLR4). AMφ did not activate STAT1 by tyrosine phosphorylation on Y701 following stimulation of any of these three TLRs, but readily did so in response to exogenous IFN-β. This unique AMφ response was not due to altered TLR expression, or defective immediate-early gene response, as measured by expression of TNF-α and three β chemokines. Instead, AMφ differed from PMφ in not producing bioactive IFN-β, as confirmed by ELISA and by the failure of supernatants from TLR-stimulated AMφ to induce STAT1 phosphorylation in PMφ. Consequently, AMφ did not produce the microbicidal effector molecule NO following TLR4 or TLR3 stimulation unless exogenous IFN-β was also added. Thus, murine AMφ respond to bacterial or viral PAMPs by producing inflammatory cytokines and chemokines, but because they lack the feed-forward amplification typically mediated by autocrine IFN-β secretion and STAT1 activation, require exogenous IFN to mount a second phase of host defense.

Cutting Edge: Antigen-Driven Lymphocyte Recruitment to the Lung Is Diminished in the Absence of Urokinase-Type Plasminogen Activator (uPA) Receptor, but Is Independent of uPA

The requirement for urokinase plasminogen activator (uPA) and uPA receptor (uPAR) in T lymphocyte migration is unknown. uPA−/− mice have fewer pulmonary lymphocytes in response to certain infections, but its unknown whether this is due to diminished recruitment. Primed, recipient mice were IT inoculated with Ag. Three days later, fluorescently labeled lymphoblasts from background-matched control wild-type (WT), uPA−/−, or uPAR−/− donor mice were injected i.v., and their recruitment was determined. Approximately twice the number of uPA−/− compared with WT lymphoblasts were recruited to the lungs of WT recipients. This difference was eliminated when uPA−/− and WT lymphoblasts were injected into uPA−/− recipients. Thus, the reduced number of lung lymphocytes in infected uPA−/− mice is not due to reduced recruitment. However, uPAR is critically involved in recruitment. Markedly fewer uPAR−/− compared with WT lymphoblasts were recruited to the lung. These findings suggest that uPAR may be a novel target for immune modulation in T lymphocyte-mediated disorders.

Recognition and phagocytosis of apoptotic T cells by resident murine tissue macrophages require multiple signal transduction events.

Macrophages (Mø) ingest apoptotic cells with unique effects on their cytokine production, but the signaling pathways involved are virtually unknown. Signal transduction in response to recognition of apoptotic thymocytes by resident murine alveolar (AMø) or peritoneal (PMø) Mø was studied by in vitro phagocytosis assay. Phagocytosis was decreased in a dose‐dependent and nontoxic manner by inhibiting phosphatidylinosiol 3 kinase (wortmannin and LY294002), protein tyrosine phosphorylation (herbimycin A, genistein, piceatannol, and for AMø only, PP2), and protein kinase C (staurosporine, Gö 6976, and calphostin C). Exposure of Mø to apoptotic or heat‐killed thymocytes, but not to viable thymocytes, activated ERK1/2 rapidly, as detected by specific phosphorylation, but did not activate NF‐κB or MAP kinases p38 or JNK. Mø phagocytosis of apoptotic T cells requires tyrosine, serine/threonine, and lipid phosphorylation. Mø recognition of apoptotic T cells triggers rapid but limited MAP kinase activation.

Repeated Intratracheal Challenge with Particulate Antigen Modulates Murine Lung Cytokines

When lungs of experimental animals are repeatedly challenged with Ag, pulmonary inflammation wanes via unknown mechanisms. We hypothesized that changes in the balance of lung cytokines are responsible for immune down-regulation to repeated Ag challenge. We used intratracheal (IT) challenge of primed C57BL/6 mice with SRBC and on various days after single (1IT) or triple (3IT) challenge counted lung inflammatory cells and measured whole-lung cytokine mRNA and protein concentrations using RT-PCR and ELISA. We found that lung lymphocyte numbers and parenchymal lung inflammation decreased significantly at days 6 and 9 after final Ag challenge in 3IT mice compared with 1IT mice. Lungs of 3IT mice showed the following changes in relative mRNA expression: an earlier peak in IL-10, decreased IL-1β, and a change from a Th2 response in 1IT mice to a Th1 response in 3IT mice (with pronounced increases in IL-12, IL-18, and IFN-γ and decreased IL-4, IL-13, and IL-5). Similar types of changes were seen in whole-lung protein concentrations for TNF-α, IL-10, IL-12 p40, IFN-γ, and IL-4. Additionally, mRNA expression of the endothelial selectins CD62E and CD62P decreased and lung lymphocyte apoptosis increased in the 3IT group. Thus, physiologic down-regulation of the pulmonary immune response to repeated Ag exposure is characterized by increased anti- and decreased proinflammatory cytokines that accompanies Th1 polarization. Similar mechanisms may act to minimize chronic lung inflammation in the majority of normal humans who do not develop progressive lung pathology when repeatedly exposed to inhaled or aspirated environmental Ags.

Subset-Specific Reductions in Lung Lymphocyte Accumulation Following Intratracheal Antigen Challenge in Endothelial Selectin-Deficient Mice

We previously demonstrated induction and expression of CD62E and CD62P in the lungs of mice primed and then challenged with intratracheal (i.t.) SRBC. The current study examined accumulation of endogenous lymphocytes in the lungs of endothelial E- and P-selectin-deficient (E−P−) mice after i.t. SRBC challenge. Compared with syngeneic wild-type (wt) mice, E−P− mice showed an 85–95% decrease in CD8+ T cells and B cells in the lungs at both early and late time points. In contrast, CD4+ T cell accumulation was reduced by ∼60% early, but equivalent to wt levels later. Surprisingly, many γδ T cells were found in lungs and blood of E−P− mice but were undetectable in the lungs and blood of wt mice. Absolute numbers of peripheral blood CD4, CD8, and B lymphocytes in E−P− mice equaled or exceeded the levels in wt mice, particularly after challenge. Trafficking studies using αβ T lymphoblasts confirmed that the recruitment of circulating cells after challenge was markedly reduced in E−P− mice. Furthermore, Ag priming occurred normally in both the selectin-deficient and wt mice, because primed lymphocytes from both groups transferred Ag sensitivity into naive wt mice. Lung production of mRNA for six CC and two CXC chemokines after challenge was equivalent by RT-PCR analysis in wt and E−P− mice. Therefore, reduced lung accumulation of αβ T cells and B cells in E−P− mice did not result from reduced delivery of circulating lymphocytes to the lungs, unsuccessful Ag priming, or defective pulmonary chemokine production. Selectin-dependent lymphocyte recruitment into the lungs following i.t.-SRBC challenge is subset specific and time dependent.

Conserved Nontypeable Haemophilus influenzae-Derived TLR2-Binding Lipopeptides Synergize with IFN-β to Increase Cytokine Production by Resident Murine and Human Alveolar Macrophages

Nontypeable Haemophilus influenzae (NTHi) is strongly associated with exacerbations of chronic obstructive pulmonary disease, which often coincide with viral respiratory infections. TLR2 contributes importantly to innate immunity to NTHi, but whether this pathway is affected by simultaneous antiviral responses is unknown. To analyze potential interactions, resident murine and human alveolar macrophages (AMφ) were exposed, in the presence or absence of the appropriate rIFN-β, to synthetic lipopeptides corresponding to the triacylated N-terminal fragments of three outer membrane proteins (OMP) (PCP, P4, and P6) that are highly conserved among different NTHi strains. Synthetic OMP elicited strong release of IL-6, the principal inducer of airway mucin genes, and induced CCL5 and CXCL10 from murine AMφ only when IFN-β was also present. Surprisingly, combined stimulation by OMPs and IFN-β also markedly enhanced TNF-α release by murine AMφ. Stimulation with PCP plus IFN-β induced IFN-regulatory factor 1 expression and sustained STAT1 activation, but did not alter the activation of MAPKs or NF-κB. AMφ derived from STAT1-deficient mice did not demonstrate increased production of TNF-α in response to PCP plus IFN-β. Analysis of wild-type and STAT1-deficient AMφ using real-time PCR showed that increased TNF-α production depended on transcriptional up-regulation, but not on mRNA stabilization. The synergistic effect of synthetic OMP and IFN-β was conserved between murine AMφ and human AMφ for IL-6, but not for TNF-α. Thus, IFN-β, which is produced by virally infected respiratory epithelial cells, converts normally innocuous NTHi OMP into potent inflammatory stimulants, but does so via different mechanisms in mice and humans.

Lymphocyte-endothelial cell adhesive interactions in lung immunity: lessons from the murine response to particulate antigen.

The adhesive interaction between lymphocytes and lung endothelial cells presents an attractive arena for the development of novel therapeutic agents to modify pathologic pulmonary immune responses. The conceptual basis for choosing molecular targets to modulate this adhesive interaction derives, in large part, from results of murine experimental model systems of the pulmonary immune response. This article reviews one such model, the response of primed C57BL/6 mice to the particulate antigen sheep erythrocytes. Novel data are presented on the effect of a blocking anti-alpha(4) integrin monoclonal antibody on lung leukocyte and lymphocyte subset accumulation after intratracheal (IT) antigen challenge. Results from this model system have indicated that lymphocytes may use either the endothelial selectins or alpha(4) integrin as independent pathways to initiate recruitment into the lungs.