Results from several laboratories have demonstrated SWCNT-driven robust inflammation, granulomatous lesions and interstitial fibrosis in the lungs of exposed animals.13–17
Complex interactions between the pathophysiologic mechanisms—inflammatory response and oxidative stress, that can synergistically amplify each other and cause enhanced pulmonary toxicity - have been revealed.18
In several recent field studies airborne concentration of nanotubes was reported as high as 53 μg/m3
/SWCNT or 430 μg/m3
Thus, under manufacturing settings, lung burdens comparable to used in our study (adjusted by lung surface area) can be achieved by workers in 2.3–19.3 years (53–430 μg/m3
, lung ventilation of 9.6 m3
deposited pulmonary fraction of 10%22
). Lifetime human lung burdens at these nanotube concentrations in the air (53 or N430μg/m3
) will be 0.57 g or 4.54 g, respectively. This will bring us to assessed exposure levels in rodents that are equivalents to 0.28 or 2.23 mg/mouse. In the current study, a bolus SWCNT delivery protocol, pharyngeal aspiration, was used to expose mice. This technique provides widespread delivery of particles throughout the lung at a single time point.23,24
It has been demonstrated that at comparable particle burdens pulmonary responses to bolus instillation reflected the pulmonary response to inhalation.25,26
There is compelling evidence that poorly soluble carbonaceous nanoparticles e.g.
nanotubes are not appreciably cleared from the lungs following intratracheal instillation or inhalation.27,28
Therefore, the doses of SWCNT utilized in the current study (40–120 μg/mouse) are relevant to the actual workplace and certainly less than those that could be achieved during life-time work exposures (8hr/d, 5 d/wk, 45 yrs).29
Our data are in line with these previous reports confirming that SWCNT are capable of inducing pulmonary inflammation. In fact, we observed a dose dependent increase in cell counts including macrophages and PMNs in BAL fluid after the animals exposure to SWCNT. Notably, SWCNT-induced inflammation facilitated the recruitment of DC to the lung tissues (), increasing chances of direct DC/SWCNT interactions. The acute phase of lung inflammation (on day 1 post exposure) was characterized by an increase of both pro-inflammatory cytokines (TNF-α, IL-6) as well as chemotactic factors (MCP1). Our assessment of pulmonary damage, measured by LDH and protein release in the BAL fluid, correlates well with the observed pulmonary inflammation (see suppl., Figures S1, S2
). Altogether, SWCNT-induced inflammation favors the recruitment of immune cells to the lung and provides additional signals for APC activation and maturation in the context of “danger signals” and a specific cytokine/chemokine environment.30
In vivo, SWCNT induced a pro-inflammatory milieu in the lungs that could lead to T cell activation and systemic immunostimulation. However, our data suggest that in SWCNT-exposed animals splenic T cell responses were suppressed ().
Several lines of evidence indicate that local inflammation following SWCNT exposure is able to modify immune responses. Recent in vivo
studies revealed the adjuvant effects of various NP administered along with a specific antigen/allergen. Increased serum levels of ovalbumin (OVA)-specific IgE, as well as elevated eosinophil counts in BAL following MWCNT or SWCNT + OVA exposure were reported.2,5,31
As for the response to infectious agents, it has been demonstrated that SWCNT and diesel exhaust particles (DEP) exposure increased the severity of Listeria monocytogenes
infection in vivo
Pulmonary exposure to MWCNT caused suppressed responses of spleen cells to mitogen stimulation, as reported by Mitchell et al.10,11
In these studies, no significant pulmonary inflammation was documented. However, TGF-β release from the lung was proposed to be the mechanism of T cell dysfunction and impaired systemic immunity. Here we provide evidence that DC might be involved in translation of immune suppressive signal(s) from the inflamed lung to peripheral lymphoid tissues modulating systemic immunity.
DC are the major bridge between the innate and adaptive immune responses. DC are APC, that are highly efficient in antigen presentation and stimulation of T lymphocytes.34
DC take-up and process antigens, migrate from peripheral tissues to lymphoid organs, present antigens, produce cytokines and express co-stimulatory molecules critical for efficient activation of T cells required for the development of adaptive immune responses. DC exist in two functionally and phenotypically distinct states, immature and mature. Immature DC are widely distributed throughout the body and occupy sentinel positions in many non-lymphoid tissues including the lung. They constantly test their environment for antigens by phagocytosis, macropinocytosis, and pinocytosis.35
Immature DC express relatively low levels of MHC class I, class II, and co-stimulatory molecules.36
After engulfing antigens and activation by proinflammatory cytokines and other signals (like bacterial LPS), immature DC differentiate into mature cells. Mature DC have a reduced potential for antigen uptake but a high capacity for antigen presentation and T cell stimulation.37,38
Notably, it was previously reported that a fraction of DC may translocate from the lung to the spleen.39
While it is unlikely that SWCNT are recognized as full antigens by APC, it is plausible that SWCNT share some similarities with bacterial/viral components and are capable of triggering pattern recognition receptors on DC thus promoting DC maturation and migration. It was previously reported9
that exposure of DC to carbon black stimulated DC maturation and T cell proliferation in vitro.
A [Gd@C(82)(OH)(22)](n) fullerene derivative was also reported to induce maturation of DC and stimulate cytokine production by DC, including IL-12p70.40
In the report by Inoue et al
SWCNT have been shown to increase the number of CD86+ cells in bone marrow-derived DC culture at 5 and 10 μg/ml doses. However, in that study in DC-T cell co-cultures, OVA-specific T cell proliferation was decreased at these SWCNT doses. In our study, the expression of CD80, CD86, CD40 or MHC class II molecules was not changed on DC following 48h SWCNT exposure in vitro.
It appears that SWCNT do not provide sufficient signals to activate DC in vitro.
Current findings are supported by data of Palomaki et al
., who also did not observe significant effects of MWCNT or SWCNT on maturation of cultured DC in vitro
In our study, exposure of DC to E. coli
LPS induced phenotypical maturation of DC (). When LPS-exposed DC were mixed with T cells we observed facilitated T cell proliferation (). Administration of LPS + SWCNT to DC did not change LPS-induced DC phenotypical maturation (). Indeed, when T cells were mixed with LPS+SWCNT treated DC we observed decreased proliferation (). Combined, these findings suggest that SWCNT do not interfere with recognition of LPS by DC. We can speculate that SWCNT exposure may intervene with antigen capture/processing and/or presentation, thereby leading to compromised DC/T cell interactions. Recently, it has been shown that SWCNTs may interfere with the cytoskeleton actin, in the absence of acute cytotoxicity.42
In this context, our previous studies have shown that SWCNT are not acutely cytotoxic to primary human macrophages but exposure to SWCNT resulted in marked suppression of the ability of macrophages to ingest apoptotic cells, a process that relies heavily on reorganization of the actin cytoskeleton.43,44
Such effects could have a negative impact on the ability of DCs to stimulate T cell proliferation. Moreover, as has been published earlier, DC could suppress T cells via
expression of B7-H1/PD-1 and CTLA-4 molecules.45
Previous reports have also implicated the induction of indoleamine 2,3-dioxygenase (IDO), IL-10, arginase, NO, SOCS, Foxo3 in mechanisms by which antigen-presenting cells may regulate T-cell responses.46–51
In our preliminary experiments, we have not seen changes in IL-10 release in DC/T cells co-cultures (data not shown). Identification of potential mechanisms of T cell suppression by SWCNT-exposed DC is a subject of our further studies.
The immune suppression observed in the current study may have significant biological relevance as a defense mechanism against unnecessary and potentially harmful cytotoxic or antibody-mediated immune responses to a “harmless”, non-proliferating pathogen – SWCNT. The T cell suppression and early granuloma formation13
concomitantly lessen the acute phase of inflammation. Involvement of DC in SWCNT-induced granulomatous lesions and fibrosis warrants further investigations. Interestingly, according to our preliminary data, the T cell suppression was sustained 2 weeks after exposure to SWCNT (not shown). Obviously, the safe immunological outcome of an exposure to a “harmless pathogen” would be a tolerogenic adaptive response. However, one cannot exclude a possible role of surfactant lipid and protein components that can strongly adhere to the SWCNT surface, and hence completely change their recognition profile by DC resulting in a different processing and subsequent antigen presentation to T cells. For example, we recently demonstrated that coating of SWCNT with anionic phospholipids - phosphatidylserine and diacylphophatidylglycerol - may markedly stimulate recognition and uptake of SWCNT by professional phagocytes, including DC.6
This may in turn affect functional responses i.e.
antigen processing and T cell presentation. Moreover, our recent studies have indicated that coating of SWCNT with components of lung surfactant impacts the ability of phagocytes to internalize SWCNT (Kagan et al
., unpublished data). Thus, the nanoparticle-modified self- or foreign proteins can be adversely recognized by APC leading to the breakage of normal self-tolerance and the development of autoimmune or allergic responses.
In conclusion, suppressed immune responsiveness following pulmonary exposure to SWCNT is likely to augment host susceptibility to infections and may also facilitate tumor progression. The detailed mechanisms by which SWCNT-exposed DC suppress T cell proliferation require further investigations.