The mechanism underlying laser-mediated immune enhancement is not well understood at present. It does not appear to be solely caused by photothermal effects, because when the skin was maintained at 42°C by a 10 × 10 × 100 mm steel bar for 2 min followed by immunization at the warm skin with OVA as previously, OVA-specific antibody production was increased by only 20% [5
]. Furthermore, immunization conducted 2 or 4 h after laser illumination, with skin temperature having returned to normal, gave rise to a similar boost as immunization did immediately after laser illumination. Russian scientists reported that illumination of the skin with a laser of a higher power and density (0.6 W and 3 W/cm2
) enhanced humoral immunity against influenza vaccine id. delivered in mice, by induction of extracellular heat-shock protein 70 production and inflammatory responses [32
]. In contrast, laser used in our study did not induce heat-shock protein or a significant inflammatory response in laser-exposed skin [5
]. We also observed little alteration in the surface expression of costimulatory molecules CD86 and CD83 or MHC class II molecule on skin DCs. We therefore consider LVA as a noninflammatory vaccine adjuvant.
However, injection of the antigen into the site of laser illumination appeared to be crucial since if the antigen was injected into a distal site, for instance, 1 cm away from the laser-illuminated site, the immune-enhancing potential decreased substantially [5
]. In accordance with this, we found that the laser illumination greatly accelerated the motility of APCs only at the areas of laser illumination. As shown in , dermal GFP+
cells, mostly DCs and macrophages, were constantly shifting, albeit slowly, and extending pseudopods, but most of them remained at original locations during a 20-min period of recording in the control (). On the contrary, the cells in the laser-treated mice showed a high migratory ability: cells leaving their original locations and a gap appearing between arrows (original locations) and the individual cells over time. OVA injection also increased migration of APCs, albeit to a lesser extent. Strikingly, a synergistic effect was observed on APC motility when OVA was administrated into the site of laser illumination (; laser + OVA). An increase in the motility of APCs is likely to promote them to survey a greater area and facilitate their antigen sampling as recognized using dendrite surveillance extension and retraction cycling habitude (dSEARCH) [33
]. The increased motility may also lead to sufficient transportation of antigen-captured DCs to the draining lymph nodes.
Laser significantly enhances dermal antigen-presenting cell motility
We postulate that brief laser illumination can transiently alter the interstitial microarchitecture, increase the tissue permeability, and permit relatively free movement of APCs in the interstitium. Indeed, upon laser illumination, dermal collagen fibers were dissociated and the interaction between DCs and surrounding tissue scaffolds was disrupted in the site of laser illumination, in sharp contrast to the well-organized microarchitecture in the control dermal connective tissue, as revealed by transmission electron microscopy (). Dissociation of APCs with the matrix proteins is expected to free their movement [34
]. The laser illumination may also enlarge pre-formed channels in the peri-lymphatic basement membrane to assist entry of APCs into the lymphatic vessel [35
]. In support of altered interstitial resistance resulting in enhanced migration of DCs by laser illumination, we found that id. injection of DCs into the site of laser illumination increased the number of DCs migrating into the draining lymph nodes by approximately 300%, when compared with injection of DCs into the control skin, irrespective of DC maturation status. We also found that modification of pulse width, frequency and peak power at 532 nm did not significantly influence laser adjuvant effects, arguing for a physical-based mechanism. Another potential mechanism for laser-mediated immune enhancement may be the ability of laser to transiently permeabilize cellular membranes by a shock wave, which augments antigen uptake by DCs [36
]. Laser treatment has been shown to increase uptake of antisense oligonucleotide by three- or 30-fold due to a laser pulse-generated high pressure [37
]. Zeira et al
. also showed that femtosecond laser sufficiently enhanced DNA delivery into cells and induced immune responses to the encoded antigen [38
]. In addition, acceleration of interstitial flow by laser illumination can greatly assist a flow of soluble antigens from the skin to the draining lymph nodes where the antigens are presented to resident DCs. In the skin, the initial lymphatic vessels are blind-end structures with wide lamina and thin walls. These initial lymphatic vessels drain excess fluid and solutes from the interstitial space and pass them to lymph nodes via lymphatic ducts. The draining process is extremely slow under normal physiological conditions but it can be increased as many as ten times by inflammation or fever-range hyperthermia [39
]. Other mechanisms may be also involved in laser-mediated immune enhancement, including enhanced mitochondrial activity of APCs, chemical releases, altered tissue pressure, and so on. Although most of these mechanisms, such as laser-induced shock wave, accelerated interstitial flow and altered mitochondrial activity, remain largely speculative and more studies are needed to confirm, it is clear that laser augments vaccination by novel and distinct mechanisms over traditional vaccine adjuvants.
Laser illumination disrupts the dense microarchitecture of dermal connective tissue