The objective of this research was to expand on our comprehensive in vitro studies of the immunological and inflammatory effects of PM exposure in allergic innate immunity by exploring the acute effects of engineered, environmental emission-source diesel PM and ambient PM instillation on pulmonary inflammation, particle uptake and immune activation of DC subsets in vivo. Thus we wished to establish the early or acute effects of PM exposure in vivo and relate this to the notion that these agents may behave as immune adjuvants in vivo. Studying DC in pulmonary immune responses to environmental PM exposure stems from our knowledge that DC are uniquely positioned through peripheral tissues such as the lung and lymphoid or nonlymphoid organs where they are specialized for the rapid uptake, endogenous processing and presentation of antigens to naïve CD4+ and CD8+ T cells. There is a paucity of data describing interactive effects as well as mechanisms affected on activation of DC by respirable pollutants.
Our data suggest that PM species, and particularly APM and DEP, did not behave as conventional adjuvants since they were immunostimulatory both in vitro [5
] and in vivo as detailed in this study. Since these environmental PM species are complex agents, it is likely that they serve as a priming and secondary stimulus in their own right by presenting to the immune system several ligands that are recognized by the innate immune system as ‘danger signals’. We have previously demonstrated a noncanonical and pro-inflammatory pathway of DC activation on exposure to PM and aeroallergens such as ragweed extract in vitro [5
]. It is hypothesized by many groups as well as work of our own that APM or DEP exhibits immune adjuvant properties [5
]. However, virtually nothing is known of the early acute inflammatory effects of PM exposure in naïve mouse models in vivo.
In the current work, we have extended our prior in vitro studies [5
] by exploring the in vivo effects of PM in a naïve mouse model of acute APM exposure. It remains unclear whether DCs are activated by acute exposure to PM or by other factors. Suggested factors contributing to DC activation include damage to the lung epithelium and oxidative stress responses [42
]. There is in vitro evidence suggesting that CBP directly induce DC maturation [30
]. Direct in vitro exposure of human and mouse DC to environmental stimuli such as APM and DEP also promoted mDC activation and a dysregulated pattern of cytokine responses from naïve CD4+ T cells [5
] and stimulated a pro-oxidative mode of primary lung DC maturation [12
]. We confirmed that both environmental APM and DEP were potent stimulators of mDC and pDC as well as proallergic immune responsiveness. By modeling the exposure of mice to APM, emission-source DEP and engineered PM species (e.g. AgP and CBP), we have integrated the cellular and molecular effects of all the bioorganic and inorganic components that constitute ambient PM. This approach crucially accounts for possible cooperative or antagonistic interactions in the experimental model.
We first assessed ways by which environmental as well as engineered PM induces lung tissue damage and activation of various innate inflammatory cells. First, evidence of lung inflammation was observed in the BALF following aspiration of both CBP and APM as defined by greater neutrophil recruitment to the lung as compared to the saline control as well as the laboratory-generated AgP and environmental DEP. While macrophage numbers in BALF remained consistent between treatments, only on acute exposure of mice to APM did we see infiltrating eosinophils. The infiltration of eosinophils to the lung just 24 h after acute exposure to only APM could be important since under normal resting conditions, eosinophils should be absent from the lung. IL-5 is known to be a key mediator in eosinophil activation, survival and recruitment [44
]. This observation of eosinophil accumulation in the lung is consistent with the ability of APM to drive enhanced secretion of IL-5 in ConA expanded primary peribronchial lymph node cultures (fig. ) and in cocultures of naïve CD4+ T cells stimulated by APM-exposed primary lung mDC (fig. ). IL-5 plays an important role in eosinophil activation which may also serve as producers of this cytokine [46
]. It is likely that the ability of PM exposure to provoke IL-5 secretion is in part responsible for the mobilization and accumulation of eosinophils in the lung, as we reported here.
We also found that, in contrast to other exposures, IL-5 secretion by DEP-exposed mDC and pDC was very similar and the reasons for this are unclear (fig. ). No increases in the Th2-type cytokines were observed following CBP exposure. Remarkably, the observed Th2-type immune activation of allogeneic CD4+ T cells induced by DEP- and APM-exposed mDCs was not associated with epithelial barrier integrity or even, in the case of DEP, with general lung inflammatory responses in our model. We do not suspect that the observed eosinophilic infiltration was associated with perturbed epithelial barrier integrity as measured by LDH release into the BALF. Marked increases in the extent of epithelial damage were only observed following engineered AgP exposure, conditions that also provoked the greatest levels of cellular damage as defined by LDH release and total protein detected in BALF. We also found that AgP, CBP and APM differentially induced activation of lung macrophages as determined by increased β-glucuronidase activity in the BALF (fig. ).
A known route of PM clearance from the lung involves endocytic and phagocytic mechanisms by macrophages [15
]. Thus PM uptake by macrophages or presumably DC may lead to their activation and subsequent release of β-glucuronidase in the lung. We also observed semiquantitative differences between the type of PM exposure and the relative frequency of macrophages that were seen to have obvious particle residues present in their cytoplasm (fig. ). We concluded that 24 h following either DEP or APM exposure, there were very few macrophages containing any particle residue in BALF macrophages.
In addition, our analyses of purified mDC indicated that only on exposure to APM was enhanced expression of the costimulatory cell-surface receptors CD54 (ICAM-1) and CD86 observed. Augmentation in the expression of costimulatory molecules of DC provides an important signal in consolidating the interaction of DC with naïve T cells that promotes both T cell proliferation and polarization of a Th1- or Th2-associated immune response. Subtle differences in DC maturation, associated with different patterns of expression of costimulatory molecules (such as CD80 and CD86), can influence the outcome of an immune response [23
]. Thus it was interesting to observe that while exposure of mice to APM augmented expression of only CD54 and CD86 by mDC, the expression by pDC of all costimulatory molecules was increased.
Stimulation of alloreactive CD4+ T cells is an important indication of an activated DC. We found that particulate-exposed mDCs stimulated CD4+ T cells more effectively than pDCs. A marked difference between the ability of lung mDC and pDC to promote T cell proliferation was seen in mice exposed to either AgP or PM as measured by BrdU incorporation in CD4+ T cells. The increases in cytokine production in the cocultures were also mainly restricted to the dominance of mDC activation. On coculture of mDC from AgP-exposed mice with CD4+ T cells, we observed profound increases in IL-12p40, IL-10 and IFN-γ production. The enhanced levels of IL-10 may explain in part the dampened neutrophilic influx in the lungs of AgP-exposed mice.
The mDC/allogeneic CD4+ T cell cocultures showed that in vivo exposure to environmental cues like DEP and APM induces mDC activation and a Th2-skewed pattern of cytokine responsiveness. This latter observation supports the hypothesis that certain PM species induce sensitization by mDC activation [30
]. Moreover, exposure of mouse models to ‘real-world’ environmental PM such as DEP and APM impart major influences on the innate and adaptive immune response. The immunostimulatory effects of ‘unpurified or unfractionated’ PM, as shown in this study, were shown at the levels of activated DC and pro-Th2 biased outcomes from ConA-stimulated primary lymph node cultures and allogeneic stimulatory cocultures of PM-exposed DC and naïve CD4+ T cells.
Also, others have shown and discussed in detail the emerging concept that immunostimulatory ‘real-world’ exposure such as to PM or aeroallergens does not necessarily drive host-protective Th1-biased responses as conventionally considered [48
]. By contrast, it is likely that such respirable immunostimulatory agonists that constitute ‘ambient real-world’ environments, may exert a greater potential to drive Th2 (or even tolerogenic) polarized immune responses [49
] than was previously considered. It is likely that transient and repeated episodic exposure to ambient PM or environmental DEP possesses the intrinsic potential to not only provide acute Th2-mediated inflammatory and mucosal immune responses but the potential to sustain Th2-mediated hypersensitivity to common allergens including ragweed, house dust mite allergen or other seasonal allergens [48
Further, several lines of evidence have proposed that bronchial epithelial cells are targeted by respirable pollutants in much the same way as the abundant DC that interdigitate those epithelial cells and ‘survey’ the external environment [51
]. It has been shown that ambient PM also induces epithelial cells to produce amphiregulin, granulocyte-macrophage colony-stimulating factor and MIP-3α [53
], that are known to induce recruitment and survival of DC. It seems likely therefore that DC will be among the first cells to sense and respond to inhaled PM, although the importance of bronchial/pulmonary epithelial cells cannot be overlooked. It is possible that exposure to PM disrupts the integrity of pulmonary epithelial cells and thereby releases inflammatory cytokines that activate DC and drive Th2-mediated allergic immunity as suggested by others [57
]. An attractive mechanism responsible for DC activation by epithelial cells suggests release of granulocyte-macrophage colony-stimulating factor and thymic stromal lymphopoietin (TSLP) on exposure to particulate pollutants that may also involve oxidative stress and the generation of reactive oxygen species in the activation of pulmonary DC [11
In the murine model at least, release of TSLP by bronchial/pulmonary epithelial cells may also instruct DC activation and secretion of Th2-driving cytokines by them, which is not surprising given the notion that TSLP conditions the lung microenvironment for Th2-mediated immune responses [60
]. However, it is likely that both the perturbation of epithelial cell barrier integrity and direct activation of DC are important in the elicitation of a proallergic innate immune response to ‘environmental danger signals’ since interdigitating DC are after all capable of sampling ambient PM via the tight junctions of pulmonary epithelia [35
]. It is also important to point out that it has been shown that relatively larger macromolecules (for example respirable coarse/fine ambient PM) may not overcome the integrity of the bronchial epithelial cell barrier and instead may be deposited and accumulated on the luminal surface of the epithelia where DC residing essentially underneath the epithelial barrier may directly capture those particles without affecting the integrity of the epithelia at all [26
We are beginning to appreciate the potential consequences of the interaction between cells of the innate immune system and exposure to PM species in driving pulmonary inflammation and Th2-mediated proallergic diseases. Our studies exploring immunological and inflammatory effects of laboratory-generated (engineered) PM and environmental ambient PM exposure in vivo are concordant with recently described work [65
]. Taken together, we have shown that exposure to APM and DEP induces in vivo mDC activation and their stimulation of naïve lymph node resident T cells and in the absence of either increased cytotoxicity or changes in lung epithelial permeability. We also reported polymorphonuclear leukocyte and eosinophilic infiltration in the lungs of APM-exposed mice without any secondary allergen challenge, an observation consistent with a proallergic response.
We conclude that ambient environmental PM exposure may directly activate lung DC, thus contributing to their translocation to the lung draining lymph nodes and subsequent provocation of a Th2-biased immune response. Our work supports the concept that environmental PM species possess selective abilities to drive Th2-biased immune responses, which is one of the central tenets of allergic respiratory diseases like asthma as suggested by others and from our prior work [3
]. We have also reported marked differences in the biological effects of the particle types studied and their differential abilities to induce Th2-biased immune responses and DC activation. Subsequent studies will be designed such that ‘real-world’ inhalation exposure models can be developed, testing both acute and repeated exposure. Attempts to study the relationship between host innate immunity and exposure to environmental respirable pollutants should enable a greater appreciation of the immune pathology of allergic diseases such as asthma and provide improved tools for the management of allergic airways disease.