Can dendritic cells see light?
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Chen, Aaron C.-H. et al. “Can dendritic cells see light?.”
Biophotonics and Immune Responses V. Ed. Wei R. Chen. San
Francisco, California, USA: SPIE, 2010. 756504-7. ©2010 SPIE-The International Society for Optical Engineering.
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http://dx.doi.org/10.1117/12.842959
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Can Dendritic Cells See Light?
Aaron C-H Chena, Ying-Ying Huang,
a,b,c
Sulbha K Sharmaa , Michael R Hamblin a,b,d,*
a Wellman Center for Photomedicine, Massachusetts General Hospital,
b Department of Dermatology, Harvard Medical School,
c Aesthetic and Plastic Center of Guangxi Medical University, Nanning, P.R China
d Harvard-MIT Division of Health Sciences and Technology,
* corresponding author: BAR414, 40 Blossom Street, Massachusetts General Hospital, Boston,
MA02114. email: Hamblin@helix.mgh.harvard.edu. Fax :617-726-8566
ABSTRACT
There are many reports showing that low-level light/laser therapy (LLLT) can enhance wound healing,
upregulate cell proliferation and has anti-apoptotic effects by activating intracellular protective genes. In
the field of immune response study, it is not known with any certainty whether light/laser is proinflammatory or anti-inflammatory. Increasingly in recent times dendritic cells have been found to play
an important role in inflammation and the immunological response. In this study, we try to look at the
impact of low level near infrared light (810-nm) on murine bone-marrow derived dendritic cells. Changes
in surface markers, including MHC II, CD80 and CD11c and the secretion of interleukins induced by light
may provide additional evidence to reveal the mystery of how light affects the maturation of dendritic
cells as well how these light-induced mature dendritic cells would affect the activation of adaptive
immune response.
Keywords: photobiomodulation, low level laser therapy, bone marrow derived dendritic cells, markers of
maturation and activation, confocal microscopy, flow cytometry
INTRODUCTION
Dendritic cells are known to be efficient stimulators of T and B-lymphocytes, and they play an important
role as antigen presenting cells (APC) in initiating and modulating immune response. Langerhans cells
were the first type of dendritic cells discovered in the skin back in 1868, but modern understanding of DC
only started about 25 years ago.
A human has about 109 Langerhans cells locating above the proliferating keratinocytes in the skin, and
most of DC remain in an immature state, characterized by the lack of migration mobility and their
inability to stimulate T cells. Although they lack co-factors for T or B lymphocyte activation, including
CD40, CD54 and CD86, they are capable of capturing antigens and expressing them in the context of
MHC class II. Only a few DC are necessary to provoke strong T-cell response. With more and more
evidence, many immune-related symptoms have revealed the importance of DC, which has been long
overlooked before.
Photobiomodulation or low-level light therapy has been proven to have many significant effects in
enhancing healing and preventing tissue death [1, 2]. Karu’s laboratory has reported that mitochondria
are a principal intracellular target of red and near-infra-red light [3], and cytochrome c oxidase is proposed
to be a photoreceptor that absorbs light as far into the infra-red as 1000-nm [4]. There have been reports
Biophotonics and Immune Responses V, edited by Wei R. Chen, Proc. of SPIE Vol. 7565,
756504 · © 2010 SPIE · CCC code: 1605-7422/10/$18 · doi: 10.1117/12.842959
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of increased cytochrome c oxidase activity after low level light [5] and by stimulating the mitochondrial
electron transport train, increased intracellular ATP after light delivery to isolated mitochondria [6] has
been observed.
Many genes have been shown to have their transcription upregulated (or down regulated) after
illumination of cells with various wavelengths and fluences of light. For instance, illumination of human
fibroblasts with 628-nm light emitting diode led to altered expression of 111 genes of 10 functional groups
[7]. Studies have shown that this one mechanism of light-mediated gene regulation is related to the
activation of the pleiotropic transcription factor NF-kB [8] probably via generation of mitochondrial
reactive oxygen species [9].
Nuclear factor kappa B (NF-kB) is a transcription factor regulating multiple gene expression and has been
shown to govern various cellular functions, including inflammatory and stress-induced responses and
survival [10]. NF-kB activation is governed by negative feedback by IkB, an inhibitor protein that binds to
NF-kB, but can undergo ubiquitination and proteasomal degradation [11] thus freeing NF-kB to
translocate to the nucleus and initiate transcription [12].
In addition, NF-kB also plays an important role in activation of inflammation. Many studies have shown
that red or near infrared light can reduce inflammatory conditions such as arthritis [13, 14] or gingival
inflammation [15].
MATERIALS AND METHODS
Bone Marrow-derived Dendritic Cells Culture
All animal procedures were approved by the Subcommittee on Research Animal Care of the
Massachusetts General Hospital and met the guidelines of the National Institutes of Health. Bone marrowderived DC were prepared from 5- to 7-weak-old male C57 mice purchased from Jackson Laboratories
(Bar Harbor, ME).
Femurs from mice were dissected, and muscle and tissue were removed. Cleaned bones were washed
twice with Hank’s Buffer Salt Solution, and placed into culture media composed of RPMI-1640 (Gibco)
with 1% penicillin–streptomycin (Cellgro), 0.1% 2-mercaptoethanol (Invitrogen), with 10% heat
inactivated fetal bovine serum (Invitrogen) (murine DC media). Bones were cut and bone marrow was
flushed with at least 5 mL of media. The bone marrow suspension was strained with a 70 mm cell strainer
(Becton Dickinson), cells collected by centrifugation at 1500 rpm for 5 min, and erythrocytes were lysed
with ammonium chloride (155 mM NH4Cl, 10 mM KHCO3, 0.1 mM EDTA). Bone marrow cell
suspension was resuspended at 1.5x105 / ml in murine DC media with 20 ng/ mL GM-CSF (Sigma). Cells
were plated at 3 ml / well in six well plates (Corning), and incubated at 37°C with 95% relative humidity
and 5% CO2. On day 3, cells were fed by exchanging half of the media with fresh murine DC media.
Purity and yield of CD3 and CD4 positive cells were inspected by flow cytometry. In addition, cell death
was quantified by propidium iodide by flow cytometry. To assure the consistency of cell population,
plates with over 85% purity and less than 10% cell death were selected for experiments. On day 8, loosely
adherent and non-adherent cells were collected and washed twice in PBS, centrifuged and resuspended at
2 x 105/mL in 60mm cell culture treated dishes (Corning), and incubated at 37°C with 95% relative
humidity and 5% CO2 over night.
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LPS treatment.
Lipopolysaccharide from Escherichia coli purified by gel chromatography (Sigma-Aldrich, St Louis, MO)
was added to DC at a concentration of 1 or 10 g/mL. Light was delivered at two time points, either
immediately after LPS or 12 hours after LPS addition.
Low Level Light Irradiation
An 810nm laser (HOYA Conbio) was selected as light source, and light irradiation time was set at 5
minutes. 30, 3 and 0.3 J/cm2 fluences were delivered at irradiances of 100 mW/cm2, 10 mW/cm2 and 1
mW/cm2 respectively to individual dishes. Light treated dishes were incubated in the incubator for 24
hours before analysis.
Cell morphology and Quantification of Protein Expressions
After 24 hour incubation after light treatment, cell morphology was monitored by using confocal
microscopy. Cells were stained with anti-murine fluorescent antibodies against MHC class II, CD80 and
CD11c (Invitrogen molecular probes) 15 minutes before microscopy. The same antibodies were used to
quantify protein expression in live cells by flow cytometry. Propidium iodide was added to distinguish
live cells from dead cells.
Statistical Analysis
All chemiluminescence readings were normalized to total protein measured by Bradford’s technique
(BCA, Pierce Biotechnology Inc.). All assays were performed in duplicate, and each sample was read
twice and took the average. Excel software was used to perform Single-Factor ANOVA to evaluate the
statistical significance of experimental results (p < 0.05).
RESULTS
Confocal Microscopy Imaging
Lipopolysaccharide is a potent stimulator for DC activation and maturation. No other cells exhibit the
shape and motility as mature dendritic cells. With proper antigen stimulation, DC display fine long
dendrites (>10 m) and the size of the cell body also enlarges from the immature stage. A fully mature
dendritic cell also displays MHCII, CD86 and CD11c on its membrane, which allows it to capture
antigens and stimulate antigen-specific T cells. The level of CD11c does not change with levels of
maturation and/or activation of DC, thus allowing it to be used as an invariant fluorescent marker of DC
purity
Many studies have shown that low level light has diminishing positive or even negative effects at higher
fluence, such as 30 J/cm2, due to its biphasic dose response [16]. This high fluence of “low-level” light
together with more usual fluences such as 3 and 0.3 J/cm2 were selected to test if bone marrow-derived
dendritic cells responded to the 810nm continuous wave laser. After 24 hours incubation after light
irradiation, which allows dendritic cells to process activation and maturation, confocal microscopy
imaging showed that the laser alone did not change either of the membrane markers expression compared
with the untreated DC (Figure 1A and B). However, if laser was delivered to the dendritic cells
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continuously incubated with lipopolysaccharide, the surface MHCII was down-regulated while CD86 was
up-regulated (Figure 1C and D. Furthermore, the dendrites of DCs with LPS plus light were more
developed than those in the LPS only group.
Dark
NIR 30J/cm2
Figure 1. Confocal microscopy of membrane protein expressions on DC, Blue, CD11c; red, MHCII and
green, CD86.
FACS analysis of membrane markers
Confocal microscopy imaging indicated that 24 hours after irradiation was a suitable time point to
distinguish changes of membrane marker expression. At the same time point, FACS analysis on the same
three markers were quantified as shown in Figure 2. Dexamethasome is a potent synthetic compound of
the glucocorticoid class of steroid hormones and acts as an anti-inflammatory and immunosuppressive
agent. FACS showed that dexamethasome down-regulated MHCII without significantly affecting
expression levels of CD81 and CD11c. On the other hand, lipopolysaccharide up-regulated both MHCII
and CD86 after 24 hours. 810nm laser results confirmed the confocal microscopy imaging data that laser
alone did not significantly change MHCII or CD86, but in the presence of LPS, 810nm laser reduced
levels of expression of membrane MHCII but increased expression of CD86.
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7000
U-
!. 6000
I
5000
C
4000
CD11c
3000
U
C
MHCII
2000
I
1000
CD86
I I
0
0
dark
dex
810
LPS0.1ig LPS0.11g+
810
Figure 2. FACS results of measurements carried out on 10,000 cells stained with appropriate fluorescent antibodies.
DISCUSSION
This study has demonstrated that for the first time that dendritic cells respond to 810nm near infrared laser
with significant morphology change and membrane marker regulation. Furthermore, in addition to
confirming previous reports and clinical trials from other laboratories that near infrared light indeed has
effects on reducing inflammation, this study also supports the fact that near infrared potentially change
DC morphology and their membrane markers of maturation, activation and antigen-presenting.
There is evidence that light acts on cells via NF-kB pathway among others and many reports have
demonstrated that light could have different impacts on different cells and tissues. The fact that near
infrared light alone did not significantly change immature DC but showed significant changes after the
addition of LPS had induced maturation might have implied that light acting on DC requires signaling
initiation of Toll-like receptors (TLR).
It has been recently discovered that there are (at least) two NF-kB activation pathways [17]. The
canonical NF-kB signaling pathway activated in response to infections (toll-like receptor signaling) and
cytokines is based on degradation of IkB inhibitors. This pathway depends on the IkB kinase (IKK),
which contains two catalytic subunits, IKK and IKK . IKK is essential for inducible IkB
phosphorylation and degradation, whereas IKK is not. IKK is involved in processing of the NF-kB2
(p100) precursor. IKK preferentially phosphorylates NF-kB2, and this activity requires its
phosphorylation by upstream kinases, one of which may be NF-kB-inducing kinase (NIK). IKK is
therefore a pivotal component of a second NF-kB activation pathway based on regulated NF-kB2
processing rather than IkB degradation.
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While light irradiation took place immediately after addition of LPS this was an early time point in the
process of maturation of dendritic cells, photo-induced signaling could be interfering with signaling from
the TLR, and this interference could determine the relative gene expression levels of different DC markers
such as MHCII and CD86.. We also tested light irradiation at the later time point after LPS stimulation,
(12 hours after LPS), and no similar changes in MHCII and CD86 as seen at early time point were
observed (data not shown). This observation led us to believe that light did have abilities to change
behaviors of DCs, but it requires early irradiation to trigger. It is at present unclear why the levels of
MHCII and CD86 moved in different directions after laser treatment. Both these markers are considered
to be upregulated during the process of DC maturation and activation. However it is likely that the overall
result of unbalancing the coordinated expression levels of MHCII and CD86 would lead to less effective
DC function, in other words to lower immune response levels to various stimuli. This may in fact be a
partial explanation of the anti-inflammatory that is known to be one of the many benefits of LLLT.
Reduced DC activation would tend to lessen the degree of inflammation produced by both the innate and
adaptive arms of the immune system in response to diverse insults and injuries. Further work is needed to
explore the determinants of LLLT on DC in various states of maturation and activation.
ACKNOWLEDGMENTS
This work was supported by NIH grant R01AI050875, Center for Integration of Medicine and Innovative
Technology (DAMD17-02-2-0006), CDMRP Program in TBI (W81XWH-09-1-0514) and Air Force
Office of Scientific Research (FA9950-04-1-0079)
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