Previously, we have shown that the inducible expression of VEGF by lung epithelial cells (9
), secreted and present in the lung tissue at physiologic (29
) levels (≥ 0.1 ng/ml) had a stimulatory effect on local DC increasing their number and activation stage based on MHCII and costimulatory molecule expression. In this work, for the first time, we provide more detailed evidence how lung VEGF expression affects local DC populations, their activation state and function.
VEGF is generated by most tumors including lung cancer (30
). The VEGF-induced DC dysfunction was proposed to be one of the mechanisms by which tumors escape immune recognition (10
). The fact that VEGF has a contrasting effect in asthma and tumor, on pulmonary and non-pulmonary DC could have several possible explanations. First of all, in cancer patients and in animal models of tumor there is already an immunosuppressive environment (31
) that itself affects DC status and possibly modulates their VEGFR expression (32
). The other explanation is that bone marrow-, lymphoid tissue-, or peripheral blood-derived DC are different from lung DC. Although this requires further detailed examination, it is currently known that the lung lacks CD8+ DC under steady state conditions (1
) but these cells are readily being detected in lymphoid tissues and peripheral blood (34
). Currently, there are six reported mouse lymphoid tissue DC subclasses, namely CD4+, CD8+, CD4+CD8+ and CD4-CD8- DC (subdivided on at least two subpopulations) and pDC (35
). Previously, two lung DC populations were clearly distinguished, mDC and pDC (1
). Currently, there are tree reported mouse lung tissue DC subclasses, namely CD11b+ mDC, pDC, and CD103+ mDC (37
). The in vitro adherence cultures of lung mature CD8- DC were reported to give a rise to CD8+ DC (19
). To clarify the DC population that is being affected by lung VEGF expression, we performed analysis of lung cell suspensions for mDC and pDC using corresponding markers (). We did not use CD103 marker to define the third lung DC population at this time. Lung VEGF expression induces the increases in both local DC populations, mDC and pDC. It is an especially interesting observation as it has been shown that these two DC populations display opposing functions in the experimental lung asthmatic conditions (21
). Whereas lung mDC are absolutely required for both acute and chronic asthmatic responses (36
) by initiating and promoting asthmatic conditions, lung pDC play a downregulatory role in this setting by inducing regulatory T cells (21
). This could be explained, in part, by their higher expression of ICOS-L (18
, ). Interestingly, increases in both mDC and pDC in the airways of asthmatic patients have been reported previously (41
) suggesting that both DC subpopulations are affected by allergen exposure and the consequences of such.
Previous studies have demonstrated that continuous long term VEGF infusions lead to the dysfunction and abnormal differentiation of DC (10
). Furthermore, the methods of cytokine delivery to the mice have different effects on DC. The use of recombinant VEGF is unpractical as it quickly disappears from circulation and does not show any significant effect on DC (10
). Interestingly, the use of a pump infusion method for the external VEGF delivery to the mice led not only to DC downregulation but also to a dramatic sequestration of lymph node tissue with GR1+ cells with eosinophilic morphology (10
). This observation further supports our conclusion that VEGF itself induces and promotes Th2 responses.
The nature of the Ag and the local cytokine milieu can modify VEGF effects on DC. A direct effect of VEGF on DC in cultures was demonstrated using human monocyte-derived DC (12
). These cells were generated with IL-4 plus GM-CSF and then matured with either LPS or a proinflammatory cytokine cocktail (TNFα, IL-1β, IL-6 and PGE2) in the presence or absence of increasing doses of recombinant VEGF. When DC maturation was induced by LPS, VEGF addition to the cultures led to the down-regulation of costimulatory molecules and the induction of apoptosis. In contrast, in DC matured by proinflammatory cytokines, no such effect was seen. The authors also demonstrated the downregulation of VEGFR-2/Flk-1 expression with DC maturation although they did not specify if LPS and proinflammatory cytokine cocktail similarly affect VEGFRs. In addition, the VEGFR expression can be differently regulated on different DC subsets by different stimuli including VEGF itself. For example, VEGF binds VEGFR1/Flt-1 expressed on hematopoietic progenitor cells and blocks activation and transcription of NF-kB in these cells (42
) leading to their apoptosis. The downregulatory effect of VEGF on the in vitro embryonic stem cell differentiation into DC was mediated by VEGFR1 but not by VEGFR2 (30
). Moreover, VEGFR2 was found to be critical for DC differentiation as its disruption dramatically decreased DC number (27
). We show here that pulmonary mDC express VEGFR1 but not VEGFR2 (). Interestingly, VEGF expression induced lung mDC activation and the simultaneous downregulation of their expression of inhibitory VEGFR1 and induction, at least on mRNA level, of stimulatory VEGFR2.
We also show here that lung VEGF expression leads to the intermediate activation of lung mDC based on the expression pattern of their costimulatory molecules (9
), chemokines () and chemokine receptors ().While selected costimulatory molecule expression on lung DC have been a subject of several studies (9
), there is only a scarce number of reports about chemokines, chemokines receptors, and other innate immunity sentinel and effector molecules regulation in lung DC.
Several in vitro
studies have shown that immature DC produce inflammatory chemokines CXCL8 (IL-8), CXCL10 (interferon-inducible protein 10, IP-10), CCL3 (MIP-1α), CCL4 (MIP-1β), and CCL5 (Regulated on Activation, Normal T Expressed and Secreted chemokine, RANTES) (23
). Upon maturation, DC loose the ability to produce these chemokines and generate T-and B-cell chemoattractants CCL17 (thymus and activation-regulated chemokine, TARC), CCL18 (DC-CK1), CCL19 (MIP-3β), and CCL22 (MDC). On the other hand, it has been shown previously that Th2-promoting DC express MCP, TARC, and secondary lymphoid organ chemokine, SLC while Th1 promoting DC express RANTES, MIP-1α, and MIP-1β (44
). Therefore, mDC in VEGF tg lungs are more Th2-promoting (). CD11b+ and CD103+ lung mDC populations were reported to be different in their chemokine production in both homeostasis and inflammation (38
) for the latter being a higher producer of Th2-promoting chemokines TARC and MDC. The regulation CD103 expression on lung mDC by VEGF remains to be determined. In addition, it has been shown that IL-12-secreting DC together with external source of IFNγ from NK cells or memory T cells prime strong Th1 response, while IL-6-secreting DC together with an external source of IL-4 prime strong Th2 response (1
). Our data on IL-6 expression by WT lung mDC () further support previous observations that lung mDC are preferential inducers of Th2 response and require an obligatory signal to induce Th1 immunity (1
). Our data on upregulation of IL-6 and IL-13 in tg lung mDC () further support our hypothesis that VEGF generates Th2-promoting lung environment through its specific action on lung DC.
Chemokine receptors expression is used as one of the characteristics to distinguish immature and mature DC (22
). Immature DC in the blood and nonpulmonary tissues express so called “proinflammatory” chemokine receptors such as CCR1, CCR2, CCR5, CCR6 and also CXCR1, CXCR2, and CXCR4 (22
). However, studies on DC in other tissues can not be extrapolated to lung DC as they are morphologically and functionally different. To this date, there are two incomplete reports on chemokine receptor expression on lung DC (20
). In one report, Swanson KA and colleagues have shown that lung immature DC express various low levels of CCR1, CCR2, CCR5, CCR6, and CCR7 (19
). Mature DC population differed from immature DC by increased expression of CCR2, CCR5 and CCR6. In other report, Chiu BC and colleagues have shown that lung DC express measurable amounts of mRNA for CCR1, CCR5, and CXCR4 while the expression of CCR2, CCR4, CCR6, CCR7, and CXCR3 was not found (47
). We show here that WT lung sorted mDC have low levels of CCR1, CCR2, CCR7, CXCR2, and CXCR4 expression (). Expression of CCR3, CCR4, CCR5, CCR6, and CCR8 was not detected. VEGF expression upregulates lung mDC expression of CCR1, CCR2, CCR7, CXCR2, and CXCR4, and induces CCR3, CCR5, and CCR8 (). This study supports our conclusion that lung mDC in VEGF tg mice are in the intermediate activation stage as, in addition to intermediate upregulation of costimulatory molecule expressions (9
), they also demonstrated upregulation of CC and CXC chemokine receptors that are found in both, immature and mature DC. In addition, upregulation of CCR7 which is a specific receptor for secondary lymphoid chemokine (SLC) and MIP-3
might, in part, explain the observed enhanced migration of lung DC into the local lymph nodes in tg mice after OVA-FITC inhalation (). Both these chemokines are constitutively expressed in afferent lymphatics and T cell area of lymph node (1
Multiple extracellular matrix degrading proteases can be upregulated and activated in DC during their migration from the blood to the lung and from the lung to the local lymph nodes. As the role of specific cathepsins in antigen processing and presentation is well established, their role in DC migration and activation is not. Multiple extracellular matrix degrading enzymes can be upregulated and activated in DC along their migration track from the blood to the lung and from the lung to the local lymph nodes. All cathepsins are able to cleave, degrade, remodel, and process extracellular matrix (49
). It is known that upon DC maturation cystein protease activity is increased (24
). We show here that lung VEGF expression leads to a strong upregulation of mRNA for cathepsins K and only marginally affects the expression of other cathepsins (). Based on our observation it is likely that mDC-derived cathepsin K is involved in mDC migration in tg mice.
MMPs play a role in the migration and maturation of DC (25
). The migration of Langerhans cells and skin DC to lymph nodes was inhibited by broad spectrum MMP inhibitor BB-3103, or by Abs to MMP-2 and MMP-9, or by administration of natural tissue inhibitors of MMPs, tissue inhibitor of matrix metalloproteases 1 and 2, TIMP-1 and TIMP-2 suggesting critical roles of these MMPs in regulating DC migration (50
). Moreover, in MMP-9 KO mice no such migration was observed, however DC maturation and their ability to stimulate T cells were intact. Interestingly, MMP-9 KO mice demonstrated significantly diminished airway inflammation, local IL-13 production, and AHR because of a defective DC migration into the airways leading to decreased levels of DC-derived Th2-attracting chemokines (25
). We show here that VEGF expression leads to the induction of MMP-9, -12, and -14 in lung mDC and upregulation of MMP-8 (). It remains critical to determine the role of cathepsins and MMPs in DC function in order to effectively manipulate it to specifically treat or prevent allergic asthmatic disease.
It is well established that fully mature lung mDC express high surface levels of MHCII and costimulatory molecules. Some Ag (43
), cytokines (51
) and other factors (17
) can lead to full DC maturation. However, certain combination of agents induces only partial lung DC activation (9
). This intermediately mature DC are unable to induce stimulation of T cells (52
). It remains to be determined if VEGF-exposed intermediately mature lung mDC are the effective T cell proliferation/cytokine production stimulators. However, the fact that we observe an asthma-like phenotype in VEGF tg mice and the reported exaggerated response of VEGF tg mice to OVA (9
) let us to hypothesize that these DC could be effective inducers of a Th2-type response. Indeed, these intermediately mature mDC display Th2-promoting phenotype based on their cytokines and chemokines expressions.
For the first time, we provide here a characteristic of WT lung mDC expression of chemokines, chemokine receptors, cytokines, cathepsins, and MMPs. This is also first observation of how VEGF affects lung mDC function. The fact that VEGF is upregulated in asthma patients (53
) provides a further proof of importance of VEGF regulation as a therapeutic strategy for asthma and other Th2 mediated disorders.