It has been shown by several groups that alveolar septal cells in the lungs from patients with severe emphysema are undergoing apoptosis [29, 33, 34], and that induction of apoptosis in mouse lungs indeed causes emphysematous changes . Alveolar septal cell apoptosis is likely the consequence of oxidative stress , withdrawal of the impact of lung cell survival factors  and/or the action of proteases . We have recently shown that alveolar septal cell apoptosis also occurs in our rat autoimmune emphysema model . Here we apply established immune modulating strategies [19, 38, 39] to demonstrate that immunity-skewing approaches can modify the autoimmune response to HUVEC immunization, reduce lung cell apoptosis and prevent airspace enlargement.
The thymus plays an important role in acquired tolerance , and several studies show that intrathymic injection of alloantigens or autoantigens induces specific systemic tolerance in experimental autoimmune models [17, 41]. The mechanism by which intrathymic injection of antigen results in systemic tolerance is still elusive, although injection of antigen into the thymus induces apoptosis of thymic T cells . In addition, an increase in clonotype positive T cells in the thymus prevents the peripheral expansion of antigen-specific CD4+ T cells  and induces prolonged anergy .
The CD4+ CD25+ regulatory population of T cells (Treg cells), which expresses the forkhead family transcription factor (Foxp3), is the key component of the peripheral tolerance mechanism that protects against a variety of autoimmune diseases . Here we demonstrate that intrathymic injection of HUVEC into 6 week old rats prior to the immunization results in the expansion of CD4+CD25+Foxp3+ Treg cells, suggesting that an expansion of this regulatory T cell subset may be one of the mechanisms responsible for the protective effect against emphysema development. Whether the mechanism of action for thymic inoculation of HUVEC is attributable to the intrathymic deletion of endothelial-reactive T cells or to the increased production of clonotype-positive T cells is the subject of ongoing investigations by our group.
In the present experiments, pristane modulated the immune response triggered by HUVEC immunization and prevented AECA production as well as lung cell apoptosis and emphysematous lung destruction. Pristane is considered as an immunological adjuvant namely in vaccines and while administration of the high doses of the hydrocarbon oil adjuvant pristane are used to induce lupus in a murine model of systemic lupus erythematosus  it also has been also shown to induce a persistent decrease in phytohemagglutinin responsiveness, consistent with a block of polyclonal T cell proliferation . As an immune adjuvant, we expected pristane to produce immune skewing. We considered that Toll-like Receptor (TLR) signaling, which is activated by pristane (e.g. requirement of TLR 7 for pristane-induced production of autoantibodies and development of murine lupus nephritis ) would be upregulated and therefore immune injury could have been ramped up in the lungs. We did not know at the outset whether pristane would exacerbate autoimmunity or ameliorate it and were surprised about the experimental results.
Similar to human emphysema , T cells recruited to the lung in response to HUVEC might express a restricted TCR repertoire, suggesting their recruitment to lung in response to a conventional antigenic stimulus. We queried whether alterations of the TCR repertoire of lung and spleen CD4+ T cells had occurred as a result of pristane treatment. Our earlier studies did not find significantly expanded TCR-Vbeta subsets in ex vivo human COPD lung samples . However, when cultured in vitro, we found seven major CD4-expressing TCR-Vbeta subset expansions from five of the patients with emphysema. This suggested that severe emphysema is associated with inflammation involving T lymphocytes that are composed of oligoclonal CD4+ T cells. Here we show that pristane treatment decreased the percentage of CD4+ T cells expressing TCR Vβ16.1 in the lung compared to the expression of Vβ16.1 on CD4+ T cells from the lungs of HUVEC-immunized rats. It has been reported that treatment with antibody directed against this receptor can facilitate prolonged graft survival , suggesting pathogenic nature of rat TCR Vβ16.1. We would like also to point out that while human and mouse TCR nomenclature is well characterized in the Immunogenetics Information System http://www.imgt.org, rat TCR are not in the system making it difficult to compare rat TCR repertoire with known nomenclatures and species.
As with VEGF receptor blockade-induced emphysema , HUVEC immunization also leads to a decrease in VEGF protein levels in the lung (Figure 5B). VEGF is likely a critical component of the lung structure maintenance program . Here we demonstrate that pretreatment with pristane abolished antibody production and preserved normal levels of VEGF in HUVEC-immunized animals. Moreover, our data indicate that HUVEC immunization induced the expansion of CD4+ T cells expressing TCR Vβ16.1, which usually are associated with an allergic response . This expansion was significantly blunted upon pre-treatment with pristane, suggesting that this T cell subset may be the pathogenic CD4+ T cell subset in this immune model of emphysema. We also found that the expression of ciliary neurotrophic factor (CNTF) was reduced in HUVEC-immunized rat lungs (Figure 5A). CNTF is a cytokine of the interleukin-6 (IL-6) family and its mRNA is widely expressed in the brain, heart, lung, and liver, of the rat, in addition to preferential expression in the sciatic nerve . To our knowledge, this is the first report that CNTF protein expression is reduced in lungs of HUVEC immunized rats, and that immunomodulation with pristane prevents this decrease.
Interleukin-1α (IL-1α) is a 19 kDa proinflammatory cytokine that is a potent mediator of the body's response to inflammation, microbial invasion, tissue injury, and immunological response , and has a role in arthritis, Alzheimer's disease and tumor growth [54, 55]. In this study, we demonstrated a significant upregulation of IL-1α levels in the HUVEC-immunized rat lung that was prevented by pristane treatment (Figure 5A).
Earlier  we have demonstrated that HUVEC injection resulted in the production of antibodies against endothelial cells. Here we show that in the lungs of HUVEC-injected rats, the number of B cells and the expression of cell surface CD19 is increased and that the immunomodulatory strategies attenuated this increase (Figure 3). Recently Sato and colleagues  reported that small changes in CD19 expression can induce autoantibody production and suggested that modest changes in the expression or function of regulatory molecules, such as CD19, may shift the balance between tolerance and immunity to autoimmunity . It is possible that downregulation of CD19 in B cells, using immunomodulatory strategies applied by us, also reflects a shift toward tolerance in our model of autoimmune emphysema. However, it is also possible that the decrease in CD19 expression could simply reflect decreased numbers of B cells in the lungs each expressing normal levels of CD19. Taken together, the results of our experiments show that rats can be made tolerant to the effects of HUVEC-immunization.
A limitation of our studies is that we have not delineated which specific cell-cell interactions, cytokines, antioxidants or proteases are involved in developing tolerance and whether the different immunomodulatory strategies converge toward a final signaling pathway. However, we can conclude that - as there are multiple factors and conditions which can trigger lung emphysema development [12, 57] - there are multiple interventions which can prevent experimental emphysema development.
Emphysema, until recently, has been seen as a problem of proteolysis, not of adaptive immunity. Whereas this is somewhat perplexing because the burning cigarette can be seen as an antigen delivery device, most recently a concept of emphysema pathogenesis based on immune mechanisms is emerging [11, 12]. Lung gene expression profiling of cigarette smoke-exposed rats demonstrated a sustained increased expression of a number of genes implicated in the innate and adaptive immune responses . Chronic lung cell damage, and in particular apoptosis when combined with ineffective phagocytosis (removal of apoptosed cell bodies) , may result in the generation of neoantigen peptides  and of autoantibodies. Moreover, tobacco anti-idiotypic antibodies have been identified in serum from smokers , and the recent finding of anti-elastin autoantibodies  in patients with tobacco smoking-induced emphysema, that correlated with severity of the disease, link emphysema to adaptive immunity against a specific lung antigen and suggest the potential for autoimmune pathology.
Whether "priming" strategies can also protect against other - non-HUVEC-induced forms of emphysema remains to be investigated. We are intrigued that experimental manipulations as diverse as intrathymic antigen injection and intraperitoneal delivery of pristane prevented the accumulation of lung B cells and macrophages, facilitated the expansion of CD4+CD25+Foxp3+ Treg cells and suppressed the expansion of TCR Vβ16.1 expressing T cells in the HUVEC-immunized animals. Whether or not TCR Vβ16.1 expressing T cells are associated with autoimmune emphysema still need to be verified. Whether or not this is true in human disease needs to be determined. Also, the role of macrophages in this autoimmune emphysema model deserves further investigation.