SummaryImmunohistochemical analysis of challenge sites such as skin and the peritoneal cavity has identified neutrophils as virtually the sole cellular participants in acute bacterial inflammation, peak influx occurring 24-48 h in advance of mononuclear cell populations associated with adaptive immunity This study challenges the general applicability of this paradigm. We demonstrate here that the earliest detectable cellular response after inhalation ofMoraxdla catarrhalis organisms is the recruitment of putative class II major histocompatibflity complex-bearing dendritic cell (DC) precursors into the airway epithelium, the initial wave arriving in advance of the neutrophil influx. Unlike the neutrophils which rapidly transit into the airway lumen, the DC precursors remain within the epithelium during the acute inflammatory response where they differentiate, and develop the dendriform morphology typical of resident DC found in the normal epithelium. During the ensuing 48-h period, these cells then migrate to the regional lymph nodes. No comparable DC response was observed after epidermal or intraperitoneal challenge, and it may be that mucosal surfaces are unique in their requirement for rapid DC responses during acute inflammation. We hypothesize that the role of the DC influx during acute inflammation may be surveiUance for opportunistic viruses, and that this covert protective mechanism is operative at a restricted number of mucosal tissue sites. p revious reports from this laboratory have established the importance of dendritic cells (DC) as APC within normal lung tissue (1, 2), and have additionally shown that a functionally and morphologically identical DC population exists within the epithelial lining of the conducting airways of both humans (3) and rodents (4, 5), where they form a contiguous network analogous to the Langerhans cell (LC) population in the epidermis.We have also recently presented evidence that the density, distribution, and surface phenotype of airway DC populations reflects the level of stimulation provided by inhalation of airborne irritant stimuli (5). Moreover, brief exposure to aerosolized bacterial LPS was demonstrated to induce a transient increase (,o50%) in the density of airway intraepithelial DC during the 24-48-h period after exposure, suggesting active participation of DC in the acute inflammatory response (5). The present study sought to further elucidate the role of DC in acute inflammation in the airways, employing a much more potent inhaled stimulus in the form of whole bacteria. Materiah and MethodsAnimals. Specific pathogen-free (SPF) adult PVG rats were used in these experiments. They were barrier housed under dust-free conditions, as detailed previously (5).Aerosol Exposure The animals were exposed for 60 rain to an aerosol of heat-killed MoraxeUa catarrhalis organisms (clinical hospital isohte) suspended in normal saline at '~109 CFU/ml.Antibodies and Iraraunostaining. The mAbs Ox6 (Ia), Ox19 (CDS; pan T cell), Ox12 (r light chains; pan B cell) (6), and Ox 42 (~ cha...

A key rate-limiting step in the adaptive immune response at peripheral challenge sites is the transmission of antigen signals to T cells in regional lymph nodes. Recent evidence suggests that specialized dendritic cells (DC) fulfill this surveillance function in the resting state, but their relatively slow turnover in most peripheral tissues brings into question their effectiveness in signaling the arrival of highly pathogenic sources of antigen which require immediate mobilization of the full range of host defenses for maintenance of homeostasis. However, the present report demonstrates that recruitment of a wave of DC into the respiratory tract mucosa is a universal feature of the acute cellular response to local challenge with bacterial, viral, and soluble protein antigens. Consistent with this finding, we also demonstrate that freshly isolated respiratory mucosal DC respond in vitro to a variety of CC chemokines as well as complementary cleavage products and N-formyl-methionyl-leucine-phenylalanine. This suggests that rapid amplification of specific antigen surveillance at peripheral challenge sites is an integral feature of the innate immune response at mucosal surfaces, and serves as an “early warning system” to alert the adaptive immune system to incoming pathogens.

Therapeutic use of IL-2 can generate antitumor immunity; however, a variety of different mechanisms have been reported. We injected IL-2 intratumorally (i.t.) at different stages of growth, using our unique murine model of mesothelioma (AE17; and AE17 transfected with secretory OVA (AE17-sOVA)), and systematically analyzed real-time events as they occurred in vivo. The majority of mice with small tumors when treatment commenced displayed complete tumor regression, remained tumor free for >2 mo, and survived rechallenge with AE17 tumor cells. However, mice with large tumors at the start of treatment failed to respond. Timing experiments showed that IL-2-mediated responses were dependent upon tumor size, not on the duration of disease. Although i.t. IL-2 did not alter tumor Ag presentation in draining lymph nodes, it did enhance a previously primed, endogenous, tumor-specific in vivo CTL response that coincided with regressing tumors. Both CD4+ and CD8+ cells were required for IL-2-mediated tumor eradication, because IL-2 therapy failed in CD4+-depleted, CD8+-depleted, and both CD4+- and CD8+-depleted C57BL/6J animals. Tumor-infiltrating CD8+ T cells, but not CD4+ T cells, increased in association with a marked reduction in tumor-associated vascularity. Destruction of blood vessels required CD8+ T cells, because this did not occur in nude mice or in CD8+-depleted C57BL/6J mice. These results show that repeated doses of i.t. (but not systemic) IL-2 mediates tumor regression via an enhanced endogenous tumor-specific CTL response concomitant with reduced vasculature, thereby demonstrating a novel mechanism for IL-2 activity.

BackgroundFunctional loss of blood–brain barrier (BBB) is suggested to be pivotal to pathogenesis and pathology of vascular-based neurodegenerative disorders such as Alzheimer’s disease. We recently reported in wild-type mice maintained on standard diets, progressive deterioration of capillary function with aging concomitant with heightened neuroinflammation. However, the mice used in this study were relatively young (12 months of age) and potential mechanisms for loss of capillary integrity were not investigated per se. The current study therefore extended the previous finding to investigate the effect of aging on BBB integrity in aged mice at 24 months and its potential underlying molecular mechanisms.ResultsImmunomicroscopy analyses confirmed significantly increased capillary permeability with heightened neuroinflammation in naturally aged 24-month old mice compared to young control at 3 months of age. Aged mice showed significant attenuation in the expression of BBB tight junction proteins, occludin-1 and to lesser extent ZO-1 compared to young mice. In addition, TNF-α in cerebral endothelial cells of aged mice was significantly elevated compared to controls and this was associated with heightened peripheral inflammation. The expression of ICAM-1 and VCAM-1 remained unelevated, and no sign of leukocyte recruitment was observed in aged mice.ConclusionThe BBB breakdown that occurs during ordinary aging is associated with inflammation and disruption of tight junction complex assembly but not through leukocyte trafficking.Electronic supplementary materialThe online version of this article (doi:10.1186/s12979-015-0029-9) contains supplementary material, which is available to authorized users.

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SummaryChanges to innate cells, such as macrophages and myeloidderived suppressor cells (MDSCs), during aging in healthy or tumor-bearing hosts are not well understood. We compared macrophage subpopulations and MDSCs from healthy young (6-8 weeks) C57BL/6J mice to those from healthy geriatric (24-28 months) mice. Spleens, lymph nodes, and bone marrow of geriatric hosts contained significantly more M2 macrophages and MDSCs than their younger counterparts. Peritoneal macrophages from geriatric, but not young, mice co-expressed CD40 and CX3CR1 that are usually mutually exclusively expressed by M1 or M2 macrophages. Nonetheless, macrophages from geriatric mice responded to M1 or M2 stimuli similarly to macrophages from young mice, although they secreted higher levels of TGF-b in response to IL-4. We mimicked conditions that may occur within tumors by exposing macrophages from young vs. geriatric mice to mesothelioma or lung carcinoma tumor cell-derived supernatants. While both supernatants skewed macrophages toward the M2-phenotype regardless of age, only geriatric-derived macrophages produced IL-4, suggesting a more immunosuppressive tumor microenvironment will be established in the elderly. Both geriatric-and young-derived macrophages induced allogeneic T-cell proliferation, regardless of the stimuli used, including tumor supernatant. However, only macrophages from young mice induced T-cell IFN-c production. We examined the potential of an IL-2/agonist anti-CD40 antibody immunotherapy that eradicates large tumors in young hosts to activate macrophages from geriatric mice. IL-2-/CD40-activated macrophages rescued T-cell production of IFN-c in geriatric mice. Therefore, targeting macrophages with IL-2/anti-CD40 antibody may improve innate and T-cell immunity in aging hosts.

SlLlnlmal'yThe relative inefficiency of respiratory mucosal immune function during infancy is generally attributed to the immaturity of the neonatal T cell system. However, immune competence in the adult lung has recently been shown to be closely linked to the functional capacity of local networks of intraepithelial dendritic cells (DC). This study examines the density and distribution of these DC throughout the neonatal respiratory tract in rats, focusing particularly on microenvironmental regulation of their class II major histocompatibility complex (MHC) (Ia) expression. In animals housed under dust-controlled conditions, airway epithelial and alveolar Ia § DC detectable by immunostaining with the monoclonal antibody (mAb) Ox6 are usually not seen until day 2-3 after birth, and adult-equivalent staining patterns are not observed until after weaning. In contrast, the mAb Ox62 detects large numbers of DC in fetal, infant, and adult rat airway epithelium. Costaining of these Ox62 § DC with Ox6 is rare in the neonate and increases progressively throughout infancy, and by weaning Ia § DC comprised, on average, 65% of the overall intraepithelial DC population. In infant rats, Ia § DC are observed first at the base of the nasal turbinates, sites of maximum exposure to inhaled particulates, suggesting that their maturation is driven in part by inflammatory stimuli. Consistent with this suggestion, densitometric analysis ofla staining intensity of individual DC demonstrates that by 2-3 d after birth, Ia expression by nasal epithelial DC was comparable with that of Iahie h epidermal Langerhans cells in adjacent facial skin, at a time when expression by tracheal epithelial DC was 7-10-fold lower. Additionally, the rate of postnatal appearance of Iahie h DC in the airway epithelium was increased by administration of interferon % and decreased by exposure of infant rats to aerosolized steroid. These findings collectively suggest that Ia expression by neonatal respiratory tract DC is locally controlled and can be upregulated by mediators that are produced within the lung and airway epithelium in response to inhalation of proinflammatory stimuli. It was also noted that Ia/~ neonatal airway DC expressed adult equivalent levels of class I MHC, which suggests differences in capacity to prime for CD8+-dependent versus CD4 § immunity to inhaled pathogens, during the early postnatal period.

Anti-cancer immunotherapies aim to generate resolution of all existing tumors, including inaccessible ones, and provide long-term protection against recurrence. This is rarely achieved. Thus, we aimed to determine if the tumor microenvironment could be turned into a potent 'self'-vaccine site. Our target was to eradicate larger tumor burdens. Our models respond to single-agent immunotherapies; however, they fail at a precisely defined 'cut-off' tumor burden. Thus, this system was used to define the immune mechanisms required to mediate regression of larger tumors that are resistant to mono-immunotherapies. We report that direct injection of IL-2 with agonist anti-CD40 antibody into the tumor bed resulted in permanent resolution of treated and untreated distal tumors. Tumor-infiltrating CD8(+) T cells and neutrophils collaborated to eradicate treated tumors, IFNgamma was not critical and protective memory was preserved. This approach relied only on tumor antigens expressed within the tumor microenvironment. It also avoided systemic toxicities, did not require chemotherapy or surgery and is clinically useful because only one tumor site has to be accessible for treatment. We conclude that provoking intra-tumoral inflammation skews the tumor microenvironment from tumorigenic to immunogenic, resulting in the resolution of treated and untreated distal tumors, as well long-term protective memory.