Critical points of tumor necrosis factor action in central nervous system autoimmune inflammation defined by gene targeting.

Tumor necrosis factor (TNF)-dependent sites of action in the generation of autoimmune inflammation have been defined by targeted disruption of TNF in the C57BL/6 mouse strain. C57BL/6 mice are susceptible to an inflammatory, demyelinating form of experimental autoimmune encephalomyelitis (EAE) induced by the 35-55 peptide of myelin oligodendrocyte glycoprotein. Direct targeting of a strain in which EAE was inducible was necessary, as the location of the TNF gene renders segregation of the mutated allele from the original major histocompatibility complex by backcrossing virtually impossible. In this way a single gene effect was studied. We show here that TNF is obligatory for normal initiation of the neurological deficit, as demonstrated by a significant (6 d) delay in disease in its absence relative to wild-type (WT) mice. During this delay, comparable numbers of leukocytes were isolated from the perfused central nervous system (CNS) of WT and TNF-/- mice. However, in the TNF-/- mice, immunohistological analysis of CNS tissue indicated that leukocytes failed to form the typical mature perivascular cuffs observed in WT mice at this same time point. Severe EAE, including paralysis and widespread CNS perivascular inflammation, eventually developed without TNF. TNF-/- and WT mice recovered from the acute illness at the same time, such that the overall disease course in TNF-/- mice was only 60% of the course in control mice. Primary demyelination occurred in both WT and TNF-/- mice, although it was of variable magnitude. These results are consistent with the TNF dependence of processes controlling initial leukocyte movement within the CNS. Nevertheless, potent alternative mechanisms exist to mediate all other phases of EAE.

Challenging cytokine redundancy: inflammatory cell movement and clinical course of experimental autoimmune encephalomyelitis are normal in lymphotoxin-deficient, but not tumor necrosis factor-deficient, mice.

Lymphotoxin (LT) is widely regarded as a proinflammatory cytokine with activities equivalent to tumor necrosis factor (TNF). The contribution of LT to experimental autoimmune encephalomyelitis (EAE) was examined using TNF/LTalpha-/- mice, TNF-/- mice, and a new LTalpha-/- line described here. All mice were generated directly in the C57BL/6 strain and used for the preparation of radiation bone marrow chimeras to reconstitute peripheral lymphoid organs and restore immunocompetence. This approach overcame the problems related to the lack of lymph nodes that results from LTalpha gene targeting. We show here that when LT is absent but TNF is present, EAE progresses normally. In contrast, when TNF is absent but LT is present, EAE is delayed in onset and inflammatory leukocytes fail to move normally into the central nervous system parenchyma, even at the peak of disease. In the absence of both cytokines, the clinical and histological picture is identical to that seen when TNF alone is deficient, including demyelination. Furthermore, the therapeutic inhibition of TNF and LTalpha with soluble TNF receptor in unmanipulated wild-type or TNF-/- mice exactly reproduces these outcomes. We conclude from these studies that TNF and LT are functionally distinct cytokines in vivo, and despite sharing common receptors, show no redundancy of function nor mutual compensation.

UV inhibits allergic airways disease in mice by reducing effector CD4 T cells.

BACKGROUND: In human asthma, and experimental allergic airways disease in mice, antigen-presenting cells and CD4(+) effector cells at the airway mucosa orchestrate, and CD4(+)CD25(+) regulatory T cells attenuate, allergen immunity. UV irradiation of skin before sensitization with ovalbumin (OVA) causes significantly reduced asthma-like responses in respiratory tissues. OBJECTIVE: To determine whether UV-induced changes in CD11c(+) cells, CD4(+)CD25(+) effector cells or CD4(+)CD25(+) regulatory cells in the trachea and airway draining lymph nodes (ADLNs) were responsible for reduced allergic airways disease. METHODS: The phenotype and function of CD11c(+) cells and CD4(+)CD25(+) cells in the trachea and ADLNs of UV- and non-irradiated, OVA-sensitized mice was examined 24 h after a single exposure to aerosolized OVA. RESULTS: No changes in the function of CD11c(+) cells from UV-irradiated mice were observed. CD4(+)CD25(+) cells from UV-irradiated, OVA-sensitized mice harvested 24 h after OVA aerosol proliferated less in response to OVA in vitro and were unable to suppress the proliferation of OVA-sensitized responder cells. This result suggested reduced activation of effector T cells in the airway mucosa of UV-irradiated, OVA-sensitized mice. To exclude regulatory cells of any type, there was similar proliferation in vivo to aerosolized OVA by CFSE-loaded, OVA-TCR-specific CD4(+) cells adoptively transferred into UV- and non-irradiated, OVA-sensitized mice. In addition, there was no difference in the expression of regulatory T cell markers (Foxp3, IL-10, TGF-beta mRNA). To examine effector T cells, ADLN cells from UV-irradiated, OVA-sensitized and -challenged mice were cultured with OVA. There was reduced expression of the early activation marker CD69 by CD4(+)CD25(+) cells, and reduced proliferation in the absence of the regulatory cytokine, IL-10. CONCLUSION: Reduced allergic airways disease in UV-irradiated mice is due to fewer effector CD4(+)CD25(+) cells in the trachea and ADLNs, and not due to UV-induced regulatory cells.

Protection against maternal infection-associated fetal growth restriction: proof-of-concept with a microbial-derived immunomodulator.

Infection-associated inflammatory stress during pregnancy is the most common cause of fetal growth restriction and/or miscarriage. Treatment strategies for protection of at-risk mothers are limited to a narrow range of vaccines, which do not cover the bulk of the common pathogens most frequently encountered. Using mouse models, we demonstrate that oral treatment during pregnancy with a microbial-derived immunomodulator (OM85), currently used clinically for attenuation of infection-associated airway inflammatory symptoms in infants-adults, markedly reduces risk for fetal loss/growth restriction resulting from maternal challenge with bacterial lipopolysaccharide or influenza. Focusing on LPS exposure, we demonstrate that the key molecular indices of maternal inflammatory stress, notably high levels of RANTES, MIP-1α, CCL2, KC, and G-CSF (granulocyte colony-stimulating factor) in gestational tissues/serum, are abrogated by OM85 pretreatment. Systems-level analyses conducted in parallel using RNASeq revealed that OM85 pretreatment selectively tunes LPS-induced activation in maternal gestational tissues for attenuated expression of TNF, IL1, and IFNG-driven proinflammatory networks, without constraining Type1-IFN-associated networks central to first-line antimicrobial defense. This study suggests that broad-spectrum protection-of-pregnancy against infection-associated inflammatory stress, without compromising capacity for efficient pathogen eradication, represents an achievable therapeutic goal.

Defective aeroallergen surveillance by airway mucosal dendritic cells as a determinant of risk for persistent airways hyper-responsiveness in experimental asthma.

A hallmark of atopic asthma is development of chronic airways hyper-responsiveness (AHR) that persists in the face of ongoing exposure to perennial aeroallergens. We investigated underlying mechanisms in sensitized rats focusing on a strain expressing the high-allergen-responder phenotype characteristic of human atopic asthmatics, and find that their high susceptibility to aeroallergen-induced persistent AHR is associated with deficiencies in the immunoregulatory and mucosal trafficking properties of inducible T-regulatory cells (iTregs). Counterintuitively, AHR susceptibility was inversely related to aeroallergen exposure level, high exposures conferring protection. We demonstrate that underlying this AHR-susceptible phenotype is reduced capacity of airway mucosal dendritic cells (AMDCs) for allergen sampling in vivo; this defect is microenvironmentally acquired, as allergen uptake by these cells in vitro is normal. Moreover, intranasal transfer of in vitro aeroallergen-loaded AMDC from naïve animals into AHR-susceptible animals during prolonged aerosol challenge markedly boosts subsequent accumulation of iTregs in the airway mucosa and rapidly resolves their chronic AHR, suggesting that compromised antigen surveillance by AMDC resulting in defective functional programming of iTreg may be causally related to AHR susceptibility.

Boosting airway T-regulatory cells by gastrointestinal stimulation as a strategy for asthma control.

The hallmark of atopic asthma is transient airways hyperresponsiveness (AHR) preceded by aeroallergen-induced Th-cell activation. This is preceded by upregulation of CD86 on resident airway dendritic cells (DCs) that normally lack competence in T-cell triggering. Moreover, AHR duration is controlled via T-regulatory (Treg) cells, which can attenuate CD86 upregulation on DC. We show that airway mucosal Treg/DC interaction represents an accessible therapeutic target for asthma control. Notably, baseline airway Treg activity in sensitized rats can be boosted by microbe-derived stimulation of the gut, resulting in enhanced capacity to control CD86 expression on airway DC triggered by aeroallergen and accelerated resolution of AHR.

Suppression of T-cell activation by pulmonary alveolar macrophages: dissociation of effects on TcR, IL-2R expression, and proliferation.

Suppression of local T-cell activation in the lower respiratory tract by resident pulmonary alveolar macrophages (PAMs) is believed to play an important role in the maintenance of local immunological homeostasis. However, the mechanism(s) by which pulmonary alveolar macrophages regulate T-cell responses are poorly characterized. The present study examines early events during the activation process in mitogen-stimulated T-cell cultures, in which proliferation was completely blocked by the presence of pulmonary alveolar macrophages. Despite inhibition of proliferation, the T-cells demonstrated normal Ca++ flux, normal modulation of surface expression of CD3 and T-cell receptor alpha/beta (TCR alpha/beta), upregulation of interleukin-2 receptors alpha and beta (IL-2R alpha and IL-2R beta), and secretion of high levels of interleukin-2 (IL-2). Thus, pulmonary alveolar macrophage regulation of T-cell activation appears to permit initial expression of effector function, but selectively inhibits further amplification of the overall T-cell response by limiting clonal expansion of the activated effector T-cell.

Alveolar macrophages from humans and rodents selectively inhibit T-cell proliferation but permit T-cell activation and cytokine secretion.

Alveolar macrophages (AM) are thought to play a key role in the regulation of immune responses within the lung. While it is well established that AM inhibit T-cell proliferation in vitro, it is unclear whether other aspects of the T-cell activation process are also inhibited. The present study demonstrates that AM from rat, mouse and human differ markedly in the potency with which they inhibit mitogen-induced T-cell proliferation, although in humans the degree of inhibition approaches that observed in the animal systems, if antigen (as opposed to mitogen) is employed as the T-cell activating agent. Rodent and human AM also differ in the mechanisms employed to achieve this inhibition; rodent AM appear to utilize reactive nitrogen intermediates, while this does not appear to be the case for human AM. Despite these differences, T cells stimulated in the presence of AM display a similar phenotype in all species examined, i.e. CD3 down-modulation, up-regulation of interleukin-2 receptor (IL-2R) expression and IL-2 production, but inability to respond to IL-2. Thus, AM appear to allow T-cell activation and expression of T-cell effector function, while selectively inhibiting T-cell proliferation.

Selective inhibition of T cell proliferation but not expression of effector function by human alveolar macrophages.

BACKGROUND: Alveolar macrophages are thought to play an important part in regulating lung immune responses. While it is clear that human alveolar macrophages suppress T cell proliferation in vitro, the mechanisms by which this is achieved are not clear, nor is it known whether alveolar macrophages also inhibit other aspects of T cell function. METHODS: Peripheral blood mononuclear cells were stimulated with phytohaemagglutinin or house dust mite allergen, and cultured with variable numbers of autologous alveolar macrophages obtained by bronchoalveolar lavage from 20 normal subjects. RESULTS: Alveolar macrophages induced a reversible inhibition of T cell proliferation in response to both mitogen and allergen stimulation, with the latter being considerably more susceptible to inhibition. This was achieved via heterogenous mechanisms, involving both soluble factors derived from alveolar macrophages and cell-cell contact. Despite inhibiting proliferation, alveolar macrophages had little or no effect on T cell calcium flux, the characteristic changes in CD3, CD2, CD28 and interleukin-2 (IL-2) receptor expression which accompany normal T cell activation, and IL-2 and interferon gamma secretion. In contrast, alveolar macrophages inhibited the tyrosine phosphorylation of proteins which may be involved in IL-2 receptor-associated signal transduction. CONCLUSIONS: The immunoregulatory properties of alveolar macrophages are relatively selective, allowing T cell activation and cytokine secretion while inhibiting T cell proliferation within the lung.

Regulation of T-cell function in lung tissue by pulmonary alveolar macrophages.

Collagenase digestion of perfused, lavaged rat lung yields a large population of CD5+ T cells, which on current evidence appear to be recently derived from the peripheral blood pool. Two-colour cytofluorographic analysis indicates that approximately 65% are CD4+ T cells, which are predominantly of the activated/memory phenotype. By limiting dilution analysis, these peripheral lung wall T cells and their airway counterparts isolated by bronchoalveolar lavage, exhibit markedly reduced capacity to proliferate by comparison to peripheral blood T cells. However, intratracheal inoculation of liposomes containing dichloro-methylene-diphosphonate at a dosage shown to eliminate the majority of resident alveolar macrophages (AM) rapidly restores the immunocompetence of these lung T-cell populations. These results are discussed in relation to recent reports that in vivo elimination of AM from rats and mice greatly amplifies immune responses to inhaled antigens, in particular T-memory cell-dependent secondary antibody responses.

Regulation of dendritic cell recruitment into resting and inflamed airway epithelium: use of alternative chemokine receptors as a function of inducing stimulus.

Dendritic cells (DC) were purified by flow cytometry from rat tracheal mucosa; they exhibited the phenotypic characteristics of immature DC including high endocytic activity, low CD80/86 expression, and in vitro responsiveness to a broad range of CC chemokines. Daily treatment of adult rats with the selective CCR1 and CCR5 antagonist Met-RANTES reduced baseline numbers of tracheal intraepithelial DC by 50-60%, and pretreatment of animals with Met-RANTES before inhalation of aerosol containing heat-killed bacteria abolished the rapid DC influx into the epithelium that occurred in untreated controls, implicating CCR1 and CCR5 and their ligands in recruitment of immature DC precursors into resting airway tissues and during acute bacterial-induced inflammation. Comparable levels of DC recruitment were observed during airway mucosal Sendai virus infection and after aerosol challenge of sensitized animals with the soluble recall Ag OVA. However, Met-RANTES did not affect these latter responses, indicating the use of alternative chemokine receptors/ligands for DC recruitment, or possibly attraction of different DC subsets, depending on the nature of the eliciting stimulus.