ARTHROPOD/HOST INTERACTION, IMMUNITY
House Dust Mite Extracts Activate Cultured Human Dermal
Endothelial Cells to Express Adhesion Molecules and
Secrete Cytokines
LARRY G. ARLIAN,1,2 B. LAUREL ELDER,3
AND
MARJORIE S. MORGAN1
J. Med. Entomol. 46(3): 595Ð604 (2009)
ABSTRACT The human skin contacts molecules from house dust mites that are ubiquitous in many
environments. These mite-derived molecules may penetrate the skin epidermis and dermis and
contact microvascular endothelial cells and inßuence their function. The purpose of this study was
to determine the response of normal human dermal microvascular endothelial cells to extracts of the
dust mites, Dermatophagoides farinae, D. pteronyssinus, and Euroglyphus maynei with and without
endotoxin (lipopolysaccharide). Endothelial cells were stimulated with mite extracts and the expression of surface molecules and the secretion of cytokines were measured in the absence and presence
of polymyxin B to bind endotoxin. All three mite extracts stimulated endothelial cells to express
intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and
E-selectin and to secrete interleukin (IL)-6, IL-8, monocyte chemoattractant protein (MCP-1), and
granulocyte/macrophage colony stimulating factor (GM-CSF). Euroglyphus mayneiÐinduced expression of all the cell surface molecules was not inhibited when the endotoxin activity in the mite extract
was inhibited. In contrast, endothelial cells challenged with D. farinae or D. pteronyssinus extract
depleted of endotoxin activity expressed only constitutive levels of ICAM-1, VCAM-1, and E-selectin.
D. farinae and E. maynei extracts depleted of endotoxin activity still induced secretion of IL-8 and
MCP-1 but at reduced levels. Only constitutive amounts of IL-6, G-CSF, and GM-CSF were secreted
in response to any of the endotoxin-depleted mite extracts. Extracts of D. farinae, D. pteronyssinus, and
E. maynei contain both endotoxins and other molecules that can stimulate expression of cell adhesion
molecules and chemokine receptors and the secretion of cytokines by normal human microvascular
endothelial cells.
KEY WORDS cytokine, chemokine, mite, endotoxin, lipopolysaccharide
House dust mites are the source of molecules that can
inßuence innate and immune responses in humans.
Some of these molecules are allergenic and can sensitize and induce allergic reactions that may manifest
as atopic dermatitis, rhinitis (nasal and ocular), or
asthma in genetically predisposed individuals. Approximately 20 of these allergens have been characterized (Thomas et al. 2007). It is likely that there are
other bioreactive molecules from house dust mites
that are not allergenic. These bioreactive allergenic
and nonallergenic molecules in extracts of house dust
mites can potentially modulate the function of many
cell types they may contact in the skin, nasal and
ocular mucus membranes, and lung epithelium. Previous research has shown that molecules in whole mite
extracts or selected allergens (e.g., Der 1 allergen)
induce the release of proinßammatory cytokines and
the expression of adhesion molecules on many differ1 Department of Biological Sciences, Wright State University, 3640
Col. Glenn Highway, Dayton, OH 45435.
2 Corresponding author, (e-mail: [email protected]).
3 Department of Pathology, Wright State University, Dayton, OH
45435.
ent cell types. Numerous studies have shown that
molecules in house dust mite extracts can effect the
function of human Þbroblasts, keratinocytes, skin microvascular postcapillary endothelial cells, dendritic
cells, macrophages, mast cells, basophils, eosinophils,
and lung epithelial cells (Machado et al. 1996, Shimizu
et al. 1998, Chen et al. 2003, Boasen et al. 2005, Wong
et al. 2006, Sohn et al. 2007, Arlian et al. 2008, Lee et
al. 2008). Thus, molecules from dust mites may contribute to or amplify immune and inßammatory pathological reactions or the course of allergic reactions.
The mechanisms used by mite molecules to modulate the function of cells in the skin, nasal and ocular
mucus membranes, lung epithelium, and others are
largely unknown. Some of these molecules are serine
and cysteine protease enzymes. Previous studies have
shown that Der p 1 (cysteine protease), Der p 3
(trypsin), Der p 6 (chymotrypsin), and Der p 9 (collagenolytic serine protease) activate lung epithelial
cells and this is mediated through cleavage of protease-activated receptors (PARs) (Sun et al. 2001,
Reed and Kita 2004, Adam et al. 2006, Kauffman et al.
2006, Liu 2007, Thomas et al. 2007). However, Kauff-
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man et al. (2006) reported that house dust mite extracts induce responses in human alveolar type II epithelial-like A549 cells that are mediated by both
protease-dependent and protease-independent signaling pathways. Der p 1 can activate airway epithelial
cells through a PAR-2Ðindependent pathway (Adam
et al. 2006).
Other in vitro studies have shown that house dust
mite extracts or speciÞc allergens affect bronchial epithelial cells (Herbert et al. 1995) and mast cells
(Stewart et al. 1994). Der p 1 can also disrupt the IgE
network by cleavage of CD23 on activated B cells
(Hewitt et al. 1995). Likewise, Der p 1 can cleave
CD25, the human T-cell interleukin-2 (IL-2) receptor,
and it may also affect the function of B cells (Schulz
et al. 1998, Shakib et al. 1998). Molecules in extracts of
Dermatophagoides farinae (Hughes) or D. pteronyssinus (Trouessart) can modulate the secretion of
growth-related oncoprotein-␣ (GRO-␣) by normal
human epidermal keratinocytes and IL-6, IL-8, monocyte chemoattractant protein (MCP-1), and macrophage colony stimulating factor (M-CSF) by normal
human dermal Þbroblasts (Arlian et al. 2008). Dust
mite proteolytic allergens induce cultured airway epithelial cells to release IL-6 and IL-8 and produce
prostaglandin E2, granulocyte/macrophage colony
stimulating factor (GM-CSF), and eotaxin (CCL11)
(King et al. 1998, Tomee et al. 1998, Knight et al. 2000,
Sun et al. 2001, Mascia et al. 2002). Human eosinophils
are activated and degranulate in response to the mite
cysteine protease allergens (Miike and Kita 2003).
Molecules in an extract of D. pteronyssinus induce
human monocytic THP-1 cells (monocyte-like) to increase expression of MCP-1, IL-6, and IL-8 (Lee et al.
2008). House dust mite extracts activate bone marrowÐ derived dendritic cells to produce IL-6 and IL-12
and to express CD40, CD80, CD86, and MHC-II molecules (Boasen et al. 2005). Der p 1 induced secretion
of IL-1␤, IL-6, IL-10, tumor necrosis factor-␣ (TNF␣),
and GM-CSF from human blood eosinophils and surface expression of CD18 and intercellular adhesion
molecule-1 (ICAM-1) (Wong et al. 2006). Basophils
from mite-sensitive asthmatics secreted IL-4 and IL-13
when stimulated with dust mite antigen (Shimizu et al.
1998). Alveolar macrophages stimulated with whole
body extract of D. farinae released IL-6, TNF␣, and
NO (Chen et al. 2003). Airway-derived epithelial cells
gave a dose-dependent increased secretion of IL-6 and
IL-8 when stimulated with house dust mite extract,
Der p 1, and Der p 5 (Kauffman et al. 2006). Thus,
there is much evidence that molecules from house
dust mites can modulate the function of cells they
contact.
Extracts of storage and house dust mites contain
molecules that modulate the secretion of proinßammatory cytokines and chemokines by cultured normal
human dermal Þbroblasts and epidermal keratinocytes (Arlian et al. 2008). Likewise, molecules in extracts of the related scabies mite inßuence the secretion of cytokines and chemokines and expression of
adhesion molecules and chemokine receptors by cultured normal human skin microvascular endothelial
Vol. 46, no. 3
cells (Elder et al. 2006). Postcapillary venule endothelial cells of the skin play a key role in regulating the
migration of inßammatory and immune cells from the
blood stream into the dermis and thus anything that
inßuences their function also inßuences the course of
pathogenesis in the skin (Steinhoff et al. 2006). The
effect that soluble molecules in extracts of the common house dust mites have on dermal microvascular
endothelial cells is unknown. The purpose of this
study was to determine the response of normal human
microvascular endothelial cells of the skin to molecules in extracts of the house dust mites D. farinae, D.
pteronyssinus, and Euroglyphus maynei (Cooreman).
Materials and Methods
House Dust Mite Extracts. Mite material was harvested from pure colonies that were maintained on a
high-protein diet at room temperature (75⬚F) and 75%
RH. Extracts were prepared from mites that migrated
to the lids of thriving cultures (live mites of all life
stages) as previously described (Arlian et al. 2008).
Brießy, aqueous whole body extracts of the house dust
mites D. farinae (DF), D. pteronyssinus (DP), and E.
maynei (EM) were prepared by extraction into endotoxin-free distilled water for 48 h at 4⬚C. After homogenization in a TenBroeck homogenizer (Fisher,
Pittsburgh, PA), extracts were centrifuged to remove
insoluble material, and the supernatants were collected and sterile-Þltered (0.22 ␮m) into sterile vials
for storage at 4⬚C. The total protein concentration of
each extract was determined using the Bradford protein assay with bovine serum albumin as standard
(Bradford 1976).
Levels of Endotoxin Present in Dust Mite Extracts.
House dust mites have previously been reported
to contain endosymbiont bacteria and endotoxins
(Trivedi et al. 2003, Valerio et al. 2005). Therefore, we
measured endotoxin levels in the mite extracts using
a chromogenic endpoint LAL (limulus amebocyte lysate) assay kit (QCL-1000; Lonza Walkersville, Walkersville, MD) according to the manufacturerÕs directions. We also prepared a set of mite extracts that were
preincubated with polymyxin B at 30 ␮g/ml (Cardoso
et al. 2007) for 18 h at 4⬚C to bind and inactivate any
endotoxin present in them.
Reagents. Biotinylated monoclonal antibodies directed against human ICAM-1, vascular cell adhesion
molecule-1 (VCAM-1), and recombinant human TNF␣
were purchased from eBioscience (San Diego, CA). The
biotinylated monoclonal antibody directed against human E-selectin, unlabeled antibodies to CXCR-1 and
CXCR-2, and all ELISA kits for cytokine detection (DuoSet kits) were purchased from R&D Systems (Minneapolis, MN). Lipopolysaccharide (LPS), polymyxin B
sulfate, aprotinin, and E-64 were purchased from SigmaAldrich (St. Louis, MO). Horseradish peroxidaseÐconjugated streptavidin (HRP-SA) and goat anti-mouse Ig
were purchased from Fisher (Pittsburgh, PA).
Endothelial Cells. Cryopreserved adult dermal microvascular endothelial cells (HMVEC-D) and all media and growth factors were purchased from Lonza.
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Fig. 1. Dose-dependent expression of adhesion molecules by human microvascular endothelial cells stimulated for 6, 12,
or 24 h with varying doses of extracts of the house dust mites D. farinae (DF), D. pteronyssinus (DP), or E. maynei (EM)
in the absence or presence of TNF␣ at 4 ng/ml. Data are presented as means of three replicate wells of cells. (Online Þgure
in color.)
Cells were cultured in Clonetics endothelial cell basal
medium-2 supplemented with EGM-2-MV growth
factors (EGM-2) at 37⬚C in 5Ð7% CO2. Cells were
plated and grown to conßuency in Costar 96-well
culture plates (Corning, Corning, NY) for subsequent
cell ELISA and cytokine secretion studies. All studies
were performed on cells between passages 7 and 10.
Cell Stimulations and Assays. Cell stimulations were
performed as previously described (Elder et al. 2006).
Brießy, at the beginning of each experimental period,
one half of the culture medium was removed from the
wells and was replaced with medium containing the
test stimuli prepared in EGM-2 to provide the desired
Þnal concentrations in the test wells with mite extract
doses based on the protein concentrations. When
present, TNF␣ was added at a Þnal concentration of 4
ng/ml alone or along with the mite extracts at varying
doses. At the end of the stimulation periods (6, 12, or
24 h), culture supernatants were removed and frozen
at ⫺80⬚C for subsequent cytokine ELISA. The expression of cellular adhesion molecules and receptors was
determined using a cellular ELISA procedure modiÞed from Yokote et al. (1993) and described previously (Elder et al. 2006). Brießy, cells were washed
and wells were blocked for 30 min at 37⬚C in 5Ð7%
CO2. The desired concentration of primary antibody
in 50 ␮l was added to each well. All incubations were
carried out for 30 min at 37⬚C in 5Ð7% CO2 with
washing between each step. Binding of biotinylated
antibodies was detected with 50 ␮l of a 1:2,000 dilution
of HRP-SA, whereas binding of nonbiotinylated antibodies was detected with 50 ␮l of a 1:1,000 dilution of
HRP-labeled goat anti-mouse antibody. After the Þnal
wash, 50 ␮l of TMB ELISA substrate (Sigma-Aldrich)
was added to each well, and the plate was incubated
for up to 20 min before color development was
stopped with the addition of 50 ␮l of 1 M sulfuric acid.
Absorbance was measured at 450 nm using a BioTek
EL800X microplate reader.
For experiments to determine the effects of endogenous endotoxin in mite extracts, extracts (100 ␮g/ml)
or control endotoxin (LPS at 250 EU/ml Þnal) was
preincubated with polymyxin B at 30 ␮g/ml (Cardoso
et al. 2007) for 18 h at 4⬚C to bind and inactivate any
endotoxin present in the extracts before 6-h cell exposure. To determine whether proteases in the mite
extracts were mediating their effects through proteolytic cleavage of PARs, we added aprotinin (serine
protease inhibitor) or E-64 (cysteine protease inhibitor) to the mite extracts at 50 ␮g/ml (Lee et al. 2008)
and used these mixtures to stimulate the cells.
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Vol. 46, no. 3
Fig. 2. Expression of chemokine receptors by human microvascular endothelial cells stimulated for 6, 12, or 24 h with
varying doses of extracts of the house dust mites D. farinae (DF), D. pteronyssinus (DP), or E. maynei (EM) in the absence
or presence of TNF␣ at 4 ng/ml. Data are presented as means of three replicate wells of cells. (Online Þgure in color.)
Data Analysis. All dose and time course experiments
were performed twice with triplicate wells for each
individual test. In one experiment, varying doses of the
three different mite extracts were tested simultaneously at each of the three times (6, 12, or 24 h). In
the other experiment, both the doses and times were
varied, and tests were performed separately for each
mite extract. The data presented here are the average
for triplicate wells of cells from the experiment in
which both the doses and times were varied.
Results
Effects Induced by Whole Mite Extracts on Expression of Cell Adhesion Molecules and Cytokine Receptors. Dermatophagoides farinae, D. pteronyssinus, and
E. maynei extracts stimulated endothelial cells to increase expression of ICAM-1 and VCAM-1 above constitutive levels in both a dose- and time-dependent
manner (Fig. 1). All three mite extracts also stimulated
the expression of E-selectin. Likewise, TNF␣ induced
increased expression of ICAM-1, VCAM-1, and E-selectin. Generally, endothelial cells co-stimulated with
both TNF␣ and any of the house dust mite extracts
expressed greater amounts of ICAM-1 and VCAM-1
compared with either any of the house dust mite
extracts alone or TNF␣ alone. D. farinae extract stimulated greater expression of ICAM-1 than either D.
pteronyssinus or E. maynei extracts.
The chemokine receptors CXCR-1 and CXCR-2
were constitutively expressed by endothelial cells
(Fig. 2). D. farinae and E. maynei extracts induced
expression of CXCR-1 and CXCR-2 above constitutive
levels. TNF␣ did not induce increased expression of
CXCR-1. Co-stimulation with TNF␣ and D. pteronyssinus, but not D. farinae or E. maynei, induced expression of CXCR-2 above the levels expressed in the
presence of either TNF␣ alone or D. pteronyssinus
alone.
Secretion of Cytokines Induced by Stimulation
With Mite Extracts. IL-8 was constitutively secreted
by endothelial cells, whereas very little IL-6, MCP-1,
or GM-CSF was constitutively produced (Fig. 3). D.
farinae, D. pteronyssinus, and E. maynei extracts induced dose- and time-dependent secretion of IL-6,
IL-8, MCP-1, and GM-CSF. TNF␣ enhanced secretion
of IL-6, IL-8, MCP-1, and GM-CSF. Co-stimulation
with the house dust mite extracts and TNF␣ considerably increased the TNF␣-induced IL-6 production
but did not effect the level of secretion of GM-CSF
induced by TNF␣. At comparable doses, D. farinae
stimulated greater secretion of IL-6, IL-8, and MCP-1
than did D. pteronyssinus. Interestingly, E. maynei extract stimulated greater secretion of IL-6 and MCP-1
than did D. pteronyssinus extract, but D. pteronyssinus
stimulated greater secretion of IL-8 than E. maynei. D.
farinae, D. pteronyssinus, and E. maynei did not stimulate secretion of IL-1␣, IL-1␤, macrophage inßammatory protein-1␣ (MIP-1␣), or eotaxin (CCL-11)
(data not shown).
Vascular endothelial growth factor (VEGF) secretion was highest after 6-h incubations and thereafter
declined over time (Fig. 4). Neither the addition of
house dust mite extracts or of TNF␣ reversed this
effect.
Effects of Mite Extracts Depleted of Endotoxin Activity on Cell Adhesion Molecule Expression and Cytokine Secretion. We measured the endotoxin levels in
our extracts and determined that the D. farinae and E.
maynei extracts contained ⬎45,000 EU/ml, whereas
the D. pteronyssinus extract had 29,400 EU/ml. Control
endotoxin (LPS) induced expression of ICAM-1,
VCAM-1, and E-selectin by endothelial cells, and this
LPS-induced expression was inhibited by polymyxin B
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Fig. 3. Dose-dependent secretion of cytokines by human microvascular endothelial cells stimulated for 6, 12, or 24 h with
varying doses of extracts of the house dust mites D. farinae (DF), D. pteronyssinus (DP), or E. maynei (EM) in the absence
or presence of TNF␣ at 4 ng/ml. Data are presented as means of three replicate wells of cells. (Online Þgure in color.)
returning to the levels expressed constitutively (Fig.
5). Endothelial cells stimulated with E. maynei extract
alone expressed ICAM-1, VCAM-1, and E-selectin,
and the E. maynei-induced expression was not inhibited by including polymyxin B in the extract to bind
and inhibit endotoxin activity. In contrast, endothelial cells stimulated with D. farinae or D. pteronyssinus extract containing polymyxin B to inhibit
any activity caused by endogenous endotoxin
showed expression of ICAM-1, VCAM-1, and Eselectin that was reduced to constitutive levels. This
indicates that for the Dermatophagoides mites, but
not for E. maynei, the stimulatory effects on adhesion molecule expression were caused by the presence of endotoxin in the extracts.
In the presence of polymyxin B, extracts of D. farinae, D. pteronyssinus, and E. maynei stimulated little or
no secretion of IL-6, G-CSF, and GM-CSF (data not
shown). However, polymyxin BÐtreated (endotoxindepleted) extracts of D. farinae and E. maynei still
stimulated secretion of IL-8 and MCP-1 above constitutive levels (Fig. 5). Control LPS by itself stimulated secretion of large amounts of IL-6, IL-8, GCSF, GM-CSF, and MCP-1, but treatment of these
solutions with polymyxin B inhibited the LPS-induced secretion of cytokines to constitutive or nearconstitutive levels.
Role of PARs. Endothelial cells stimulated with the
mite extracts in the presence of either aprotinin
(serine protease inhibitor) or E-64 (cysteine protease
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Vol. 46, no. 3
Fig. 4. VEGF secretion by human microvascular endothelial cells stimulated for 6, 12, or 24 h with varying doses of extracts
of the house dust mites D. farinae (DF), D. pteronyssinus (DP), or E. maynei (EM) in the absence or presence of TNF␣ at
4 ng/ml. Data are presented as means of three replicate wells of cells. (Online Þgure in color.)
inhibitor) did not exhibit decreased expression of adhesion molecules or cytokine secretion, indicating
that proteolytic cleavage of PARs was not responsible
for the induction of these effects (data not shown).
Discussion
The house dust mites D. farinae, D. pteronyssinus,
and to a lesser extent E. maynei are important sources
of potent allergens in homes throughout the world.
The human skin regularly contacts house dust mites
and molecules from house dust mites that are ubiquitous in human dwellings in fabrics of soft furniture
(couches and recliners), beds (mattress, pillows and
bedding), carpets, clothing, automobile seats, and in
public places (seats in theaters, buses, trains and airplanes). Dust mites and their allergens have been
recovered from clothing and human skin (Naspitz et
al. 1997, Riley et al. 1998, Siebers et al. 1998, Neal et al.
2002, Yasueda et al. 2003). Disturbance of the epidermis and properties of some of the mite molecules
would allow mite molecules to penetrate the epidermis and dermis and contact resident (e.g., keratinocytes, Þbroblasts, and endothelial cells of the microvasculature) and migratory (e.g., macrophages, mast
cells, Langerhans cells, lymphocytes and other leukocytes) cells in the skin. House dust mite extracts contain many enzymes that may disrupt the skin barrier
function and facilitate the penetration of molecules
from mites through the skin (Cork et al. 2006). Physical abrasions, soaps and detergents, skin care products, solvents, cleaning products, and volatile organic
compounds can also disrupt the skin barrier and facilitate penetration of mite molecules (Cork et al.
2006, Huss-Marp et al. 2006). Positive patch tests are
direct evidence that dust mite molecules can penetrate the epidermis and dermis (Deleuran et al. 1998,
Yasueda et al. 2003, Darsow et al. 2004, Fuiano et al.
2008).
Previous studies found that extracts of storage and
house dust mites, as well as the related scabies mites,
inßuence the release of some proinßammatory cytokines and chemokines from epidermal keratinocytes
and dermal Þbroblasts and monocytic THP-1 cells
(Arlian et al. 2003, 2008; Lee et al. 2008). It has been
shown that Der p 1 induced expression of ICAM-1 and
VCAM-1 on human umbilical vein endothelial cells
and that Der p 2 moderately upregulated ICAM-1
but not ICAM-2 (Mastrandrea et al. 2003). Likewise,
molecules in an extract of the related mite Sarcoptes
scabiei (De Geer) can inßuence the expression of
VCAM-1, E-selectin, CXCR-1, and CXCR-2 on the
surface of dermal endothelial cells and the secretion
of IL-6 and IL-8 by these same cells (Elder et al. 2006).
In this study, we investigated the effect of D. farinae,
D. pteronyssinus, and E. maynei on human microvascular endothelial cells because these cells regulate the
extravasation of inßammatory and immune cells and
permeability of ßuids (leakage and edema) into the
dermis. The important Þnding is that molecules from
D. farinae, D. pteronyssinus, and E. maynei extracts
directly induced dose- and time-dependent expression of the adhesion molecules ICAM-1, VCAM-1, and
E-selectin by normal human endothelial cells. Likewise, D. farinae, D. pteronyssinus, and E. maynei extracts induced secretion of IL-6, IL-8, MCP-1, and
GM-CSF. IL-6 is a general promoter of inßammation
and activates other cell types in the skin. IL-8 is chemotaxic for neutrophils. MCP-1 (CCL2) functions include attracting monocytes and eosinophils (Dunzendorfer et al. 2001) and provoking the aggregation of
mast cells (Conti et al. 1995). GM-CSF locally may
activate leukocytes at sites of inßammation. Therefore, these mites are the source of molecules that
promote inßammation by stimulation of skin endothelial cells.
Human dermal microvascular endothelial cells express toll-like receptors (TLRs) (Faure et al. 2000,
Henneke and Golenbock 2002, Fitzner et al. 2008).
These cells constitutively express TLR4 that is activated by LPS and LPS may stimulate increased expression of TLR2 that is not normally constitutively
expressed by these endothelial cells (Henneke and
Golenbock 2002). Previous studies have shown that
extracts of D. farinae and D. pteronyssinus mites contain endotoxins, D. farinae much more so than D.
pteronyssinus (Michel et al. 1996, Trivedi et al. 2003,
Valerio et al. 2005) and house dust mite extracts elicit
TLR-dependent dendritic cell responses (Boasen et
al. 2005). It was possible that endotoxins in the mite
extracts activated TLRs (particularly TLR4) and this
induced the upregulated expression of cellular adhesion molecules and secreted cytokines that was observed. Therefore, we preincubated our D. farinae, D.
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Fig. 5. Effect of endotoxin inactivation by polymyxin B on mite-induced adhesion molecule expression and cytokine
secretion by human microvascular endothelial cells. Cells were stimulated for 6 h with D. farinae (DF), D. pteronyssinus (DP),
or E. maynei (EM) extract (100 ␮g/ml) or endotoxin control (LPS at 250 EU/ml) in the absence or presence of polymyxin
B (30 ␮g/ml) to bind and inactivate endotoxin. Data are presented as mean ⫾ SEM of three replicate wells of cells.
pteronyssinus, and E. maynei extracts with polymyxin
B to bind and inactivate the LPS in these extracts
(Cardoso et al. 2007, Fitzner et al. 2008). However,
using LPS-depleted extracts, our current studies
showed that the effect induced by the mite extracts
was not entirely induced by LPS in the extracts. In this
study, LPS alone induced the secretion of IL-6, IL-8,
G-CSF, GM-CSF, and MCP-1, and polymyxin B inhibited this LPS-induced effect. Extracts of D. farinae,
D. pteronyssinus, and E. maynei also induced secretion
of IL-6, IL-8, G-CSF, GM-CSF, and MCP-1. Cells stimulated with D. farinae, D. pteronyssinus, or E. maynei
in the presence of polymyxin B to inactivate endotoxin
continued to secrete only constitutive levels of IL-6,
G-CSF, and GM-CSF. However, D. farinae and E.
maynei extracts treated with polymyxin B still secreted
elevated levels of IL-8 and MCP-1, whereas only E.
maynei--stimulated cells expressed ICAM-1, VCAM-1,
and E-selectin at elevated levels. Taken together,
these results indicate that these house dust mites induced effects on endothelial cells that were not entirely induced by endotoxin acting through TLR4 and
therefore there are molecules in mite extracts that
must induce their effects by other receptors and signaling pathways.
It is interesting that extracts of D. farinae, D. pteronyssinus, and E. maynei did not stimulate endothelial
cells to the same degree despite the fact the extracts
were formulated identically and that equal protein
doses were tested. Overall, D. farinae extract had
greater stimulating ability than extracts of D. pteronyssinus or E. maynei, but in some cases, E. maynei had
greater stimulating ability than D. pteronyssinus. Although the mechanism for this is unknown, it suggests
that extracts from the different species may have different molecules in them or that they have different
concentration/amounts of similar molecules that can
modulate cytokine and adhesion molecule expression.
Our D. farinae and E. maynei extracts contained higher
levels of endotoxin than D. pteronyssinus and this may
be a contributing factor.
The reaction by endothelial cells to house dust
mites is likely caused by bioreactive molecules present
in these extracts that are both allergenic and nonal-
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lergenic. In some cases, the bioreactive molecules may
be enzymes. Any of these molecules may inßuence
endothelial cell function as well as many other cells in
the skin and the blood that are involved in inßammation and immune reactions. Previous studies using
lung epithelial cells indicate that allergenic enzymes
of dust mites can exert an effect by activation of PARs
(Reed and Kita 2004). Likewise, endothelial cells may
express PARs that are cleaved and activated by enzymes in an extract of house dust mites. Although
house dust mites are the source of some protease
allergens, we have not previously detected serine protease activity in our dust mite extracts (Morgan and
Arlian 2006). When we attempted to inhibit the proteases in the extracts used here, the expression of
cellular adhesion molecules and secretion of cytokines
was not reduced compared with stimulation with mite
extracts alone. These results suggested that D. farinae,
D. pteronyssinus, and E. maynei modulate endothelial
cells by serine and cysteine protease-independent
mechanisms and not by proteolytic cleavage of PARs.
The processes by which dermal endothelial cells
regulate cell extravasation is mediated by cytokines
and chemokines from other cells in the vicinity (e.g.,
keratinocytes, Þbroblasts, and macrophages). Inßammation in the skin involves the participation of many
different cell types. We previously showed that dust
and storage mites induce keratinocytes and Þbroblasts
to release multiple proinßammatory cytokines, some
of which are known to stimulate endothelial cells
(Arlian et al. 2008). D. farinae, D. pteronyssinus, and E.
maynei can induce secretion of growth-related oncoprotein-␣ (GRO␣) from normal human epidermal
keratinocytes and IL-8 from normal dermal Þbroblasts. D. farinae can also induce IL-6, MCP-1, and
M-CSF secretion from Þbroblasts, whereas D. pteronyssinus induces M-CSF secretion (Arlian et al. 2008).
Thus, in vivo, there is simultaneous co-stimulation of
many cell types. How the multiple mite-induced effects from Þbroblasts, keratinocytes, and endothelial
cells collectively modulate and orchestrate the response to these mites in vivo in the skin is not clear.
In vivo, the effects of mites on several other cell types
along with the direct effects of mites on endothelial
cells could together orchestrate endothelial cell function. In the culture system used here, we could not
determine the effects contributed by other cell types
that are also stimulated by house dust mite extract.
In this study, we showed that D. farinae, D. pteronyssinus, and E. maynei extracts contain molecules that
are potent stimulators of skin endothelial cells. Some
of these effects are likely caused by endotoxin present
in the extracts and mediated through TLR4, whereas
the mechanisms responsible for the other responses
are not yet clear. It does not seem that the effects of
these mite extracts are signiÞcantly mediated through
PARs.
The expression of cellular adhesion molecules and
release of cytokines and chemokines induced by D.
farinae, D. pteronyssinus, and E. maynei have signiÞcant implications in understanding the immunopathology of allergic reactions associated with atopic
Vol. 46, no. 3
dermatitis and local inßammation in the skin. This
study and our previous work (Arlian et al. 2008)
clearly show that house dust mites can provide exogenous inßammatory stimuli in the skin to Þbroblasts,
keratinocytes, and endothelial cells, and these cells
can produce a milieu of proinßammatory cytokines or
express cellular adhesion molecules in response to
mite molecules. Although these were in vitro studies,
our results suggest that these mites can induce a similar response in vivo and contribute to inßammation in
the skin should molecules penetrate the epidermis and
dermis.
Acknowledgments
This research was partially supported by a grant from the
National Institutes of Health, National Institute of Allergy
and Infectious Disease (AI-017252) to L.G.A. We thank D. L.
Vyszenski-Moher for culturing the mites.
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Received 24 November 2008; accepted 17 February 2009.