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Blood, 1 May 2001, Vol. 97, No. 9, pp. 2727-2733
IMMUNOBIOLOGY
Fever-range hyperthermia dynamically regulates lymphocyte
delivery to high endothelial venules
Sharon S. Evans,
Wan-Chao Wang,
Mark D. Bain,
Randy Burd,
Julie R. Ostberg, and
Elizabeth A. Repasky
From the Department of Immunology, Roswell Park Cancer
Institute, Carlton and Elm Streets, Buffalo, NY.
 |
Abstract |
Fever is associated with increased survival during acute
infection, although its mechanism of action is largely unknown. This study found evidence of an unexpectedly integrated mechanism by which
fever-range temperatures stimulate lymphocyte homing to secondary
lymphoid tissues by increasing L-selectin and  4 7
integrin-dependent adhesive interactions between circulating
lymphocytes and specialized high endothelial venules (HEV). Exposure of
splenic lymphocytes in vivo to fever-like whole-body hyperthermia (WBH;
39.8 ± 0.2°C for 6 hours) stimulated both L-selectin and
47 integrin-dependent adhesion of lymphocytes to HEV under shear
conditions in lymph nodes and Peyer patches. The adhesiveness of HEV
ligands for L-selectin and 47 integrin (ie, peripheral lymph node
addressin and mucosal addressin cell adhesion molecule-1) also
increased during WBH or febrile responses associated with
lipopolysaccharide-induced or turpentine-induced inflammation. Similar
increases in HEV adhesion occurred during hyperthermia treatment of
lymph node and Peyer patch organ cultures in vitro, indicating that the
local lymphoid tissue microenvironment is sufficient for the
hyperthermia response. In contrast, WBH did not augment adhesion in
squamous endothelium of nonlymphoid tissues. Analysis of homing of
47hi L-selectinlo murine TK1 cells and
L-selectinhi 47 integrin-negative 300.19/L-selectin
transfectant cells showed that fever-range temperatures caused a 3- to
4-fold increase in L-selectin and 47
integrin-dependent trafficking to secondary lymphoid tissues. Thus,
enhanced lymphocyte delivery to HEV by febrile temperatures
through bimodal regulation of lymphocyte and endothelial adhesion
provides a novel mechanism to promote immune surveillance.
(Blood. 2001;97:2727-2733)
© 2001 by The American Society of Hematology.
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Introduction |
Systemic fever and local increases in temperature
at sites of inflammation are cardinal features of host responses to
pathogenic stimuli. Although the highly conserved fever response is
linked to increased survival,1,2 the mechanisms underlying
the protective action of fever have not been fully elucidated. A
central locus of control of the host immune response to foreign
pathogens resides at the leukocyte-endothelial interface. To fight
infections in peripheral tissues, blood-borne lymphocytes gain entry
across specialized high endothelial venules (HEV) in secondary lymphoid organs (ie, lymph nodes [LN] and Peyer patches [PP]) and at
extralymphoid sites of infection. Lymphocyte adhesion to HEV is
initiated by the L-selectin and 47 integrin adhesion molecules on
the microvillous processes of lymphocytes.3,4 These
molecules mediate the initial attachment and slow rolling of
lymphocytes on HEV counterreceptors under hemodynamic shear conditions.
Subsequent G-protein-dependent chemokine activation of a
2-integrin, lymphocyte function-associated antigen 1 (LFA-1),
results in firm adhesion of lymphocytes to HEV and transendothelial
migration. Lymphocyte-HEV interactions in LN are initiated exclusively
by L-selectin recognition of sialomucin-like receptors termed
peripheral lymph node addressins (PNAd), which are identified by the
MECA-79 monoclonal antibody (mAb).3-6 In PP,
L-selectin and 47 integrins initiate lymphocyte tethering on HEV
through interactions with distinct domains of MECA-367 mAb-reactive
mucosal addressin cell adhesion molecule-1
(MAdCAM-1).3,7
Mammals respond to natural infection or inflammatory stimuli (eg,
pyrogenic cytokines, bacterial lipopolysaccharide, and turpentine) with
a mild to moderate fever (1-4°C above normal body
temperature).1,2,8 Fever-range temperatures are associated
with enhancement of the innate and adaptive arms of the immune response
through augmentation of T-cell proliferation and cytotoxicity,
bioactivity of inflammatory cytokines (eg, interferon (IFN)-), and
neutrophil motility and chemotaxis.1,2 Recent studies
suggest that the highly efficient adhesion mechanism for lymphocyte
recirculation can also be amplified by fever-range hyperthermia. In
this regard, direct exposure of human and murine lymphocytes to
long-duration, fever-range temperatures in vitro markedly stimulates
both L-selectin and 47 integrin-dependent adhesion to HEV in
frozen-section assays.9-12 In sharp contrast, fever-range
temperatures did not increase LFA-1 binding activity9 or
47 integrin-mediated adhesion to the extracellular matrix protein fibronectin.11 Hyperthermia increases L-selectin
and 47 integrin adhesion through the release of soluble autocrine factors, without affecting the surface density of these
molecules.9-12 The 11-amino-acid carboxy-terminal
cytoplasmic domain of L-selectin is required for hyperthermia-enhanced
L-selectin adhesion to HEV and hyperthermia-induced stable associations
with the detergent-insoluble cytoskeletal matrix.10 Thus,
hyperthermia-induced anchoring of L-selectin to the structural
cytoskeleton has been proposed to increase the tensile strength of
L-selectin,10 allowing adherent lymphocytes to better
withstand shear conditions in blood vessels. The role of febrile
temperatures in regulating adhesion in HEV has not been previously investigated.
In this study, we examined the possible role of fever-like whole-body
hyperthermia (WBH), which mimics the thermal element of a febrile
response, in promoting homing to lymphoid tissues through the
regulation of lymphocyte-endothelial adhesion in vivo. Experimental
approaches that raise core body temperatures to the febrile range
support investigation of direct thermal effects on adhesion, segregated
from the complex neuronal, hormonal, and cytokine networks operating as
a consequence of natural infection.1,2 We found that
fever-range WBH induced marked changes in lymphocyte distribution that
correlated with enhanced adhesion in 2 distinct cellular targets.
Specifically, WBH stimulated L-selectin and 47 integrin function
in lymphocytes as well as PNAd and MAdCAM-1 adhesion in lymphoid tissue
HEV. Similar increases in lymph node HEV adhesion were observed after
fever induction by bacterial lipopolysaccharide (LPS) stimulation or
injection of turpentine. These results provide insight into the
immunoprotective mechanisms by which febrile temperatures dynamically
modulate regional recruitment of circulating lymphocytes to tissues
during physiologic responses to infection and inflammation.
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Materials and methods |
Cells and cell lines
Human peripheral blood lymphocytes (PBL) were isolated from
healthy-donor buffy-coat leukocyte concentrates (American Red Cross,
Buffalo, NY) by Ficoll-Hypaque centrifugation.9,10 PBL
were cultured in complete medium (RPMI 1640 medium [Gibco BRL, Grand
Island, NY] with 10% fetal calf serum [Gibco BRL], 2 mM
L-glutamine, 100 U/mL penicillin, and 50 µg/mL
streptomycin). Stably transfected mouse pre-B 300.19 cell
lines10,13 (kindly provided by G. S. Kansas, Northwestern
University, Chicago, IL) that expressed either full-length human
L-selectin complementary DNA (300.19/L-selectin cells) or a deletion
mutant lacking the 11 carboxy-terminal residues of the cytoplasmic
domain (300.19/L cyto cells) were maintained in complete medium
containing 7 × 10 6 M -mercaptoethanol. The mouse
TK1 lymphoma cell line11,14,15 (a generous gift from
E. C. Butcher, Stanford University, Stanford, CA) was
maintained in complete medium containing 5.7 × 105 M
-mercaptoethanol. Cultured cells were exposed to hyperthermia (40°C) in a 5% carbon dioxide incubator.
Reagents and mAbs
DATK32 mAb (rat IgG2a; Pharmingen, San Diego, CA) recognizes a
combinatorial epitope on the mouse 47 integrin heterodimer. The
mAbs directed against mouse and human L-selectin (Mel-14.D54, rat
immunoglobulin G2a [IgG2a]; and DREG-56, mouse IgG1, respectively), MAdCAM-1 (MECA-367, rat IgG2a), and PNAd (MECA-79, rat IgM) were from
the American Type Culture Collection (Rockville, MD). Isotype-matched control antibodies were from Pharmingen. Fluorescein isothiocyanate (FITC) conjugated goat F(Ab')2 antirat IgG was from Caltag
(South San Francisco, CA).
WBH treatment and fever induction by LPS and turpentine
Female BALB/c mice (8- to 10-weeks old; Taconic Lab, Germantown,
NY) were housed at a temperature of 24 ± 0.1°C. Core temperatures of sentinel mice in all experimental groups were continuously monitored
using a subcutaneously implanted microchip thermotransponder (14 mm × 2.2 mm; implanted  1 week before experiments began) and
a programmable data-acquisition system (Biomedic Data Systems, Maywood,
NJ). The core temperature of WBH-treated mice was maintained at
39.8 ± 0.2°C by placing the animals in a 38.8°C environmental chamber (model BE5000; Memmert, East Roy, WI).16-19 In
selected experiments, whole-body temperatures were maintained at
38 ± 0.3°C, 39 ± 0.4°C, or 40 ± 0.2°C by placing mice in
an environmental chamber adjusted to 35 to 35.7°C, 35 to 36.5°C,
and 37.6 to 38.5°C, respectively. Normothermic control mice (core
temperature, 36.5 ± 0.5°C) were kept at room temperature in a
darkened cabinet for the experimental period. Sterile inflammatory
responses were induced by subcutaneous injection of 100 µL turpentine
oil (Sigma, St Louis, MO) or saline (control) into both hind
legs.8,20 LPS (10 µg/25 g body weight in 1 mL sterile
phosphate-buffed saline [PBS], Escherichia coli serotype
0127:B8; Sigma) or control saline was injected intraperitoneally into
mice as described previously.19
Lymphoid tissue sampling and flow cytometric analysis
Blood was aspirated from the retro-orbital venous plexus of
anesthetized mice and depleted of erythrocytes by means of ammonium chloride lysis. Single-cell suspensions of spleen and peripheral lymph
nodes (PLN; pooled superficial inguinal, brachial, axillary, sciatic,
superficial, and deep cervical nodes) were prepared by passing tissues
through a wire mesh and depleting erythrocytes by means of ammonium
chloride lysis. Total numbers of lymphocytes were counted by using a
hemocytometer. L-selectin and 47 integrin expression was
determined by flow cytometry as described previously.9-11 A total of 10 000 events were collected by using a FACScan flow cytometer (Becton Dickinson, Sunnyvale, CA) and analyzed with Winlist
4.0 (Verity Software House, Topsham, ME).
Frozen-section adhesion assay
Lymphocyte adhesion to HEV was evaluated in a frozen-section
adhesion assay as described previously.9-11 A total of
5 × 106 cells were overlaid on 12-µm cryosections of
PLN, PP, or pancreas tissues from WBH-treated or nontreated BALB/c
mice. Selected lymphoid tissue specimens were blocked with mAb specific
for PNAd or MAdCAM-1. Alternatively, lymphocyte samples were blocked
with mAb specific for L-selectin or 47 integrin. The assay was
performed at 4°C for 30 minutes with mechanical rotation (95-112 rpm;
Labline Instrument, Melrose Park, IL). After removal of nonadherent
cells, sections were fixed in 3% glutaraldehyde and stained with 0.5%
toluidine and absolute ethanol. Lymphocyte adhesion was quantified
using light microscopy to assess a total of 300 to 500 HEV per lymphoid tissue sample or in squamous vessels of pancreatic tissues in 10 high-powered fields per sample (equivalent area of 0.65 mm2/field).
Short-term migration assays
The 300.19/L-selectin cells and TK1 cells were cultured in vitro
at 37°C or 40°C for 6 hours and then labeled with PKH-26 fluorescent dye (Sigma) for short-term homing studies as described previously.9 Internal standard cells (ie, 300.19/L-cyto
cells or TK1 cells treated with DATK32 mAb [10 µg/mL]) were
maintained at 37°C before labeling with FITC.9
Approximately 3 × 107 PKH-26-labeled test cells were
mixed with an equal number of FITC-labeled internal standard cells and
injected into the tail vein of normothermic control BALB/c mice or
WBH-treated mice. Mice were kept at room temperature during the 1-hour
homing assay. Lymphoid tissues were then collected, dissociated into
single-cell suspensions, resuspended in equivalent volumes of 1%
formaldehyde and PBS (0.1 mL for all tissues except spleen, for which
0.3 mL was used), and mounted on glass slides. In each experiment,
PKH-26-labeled and FITC-labeled cells were quantified using
fluorescence microscopy in 10 randomly chosen 20 × fields
(equivalent area, 0.5 mm2/field) for 2 aliquots per
tissue sample.
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Results |
Fever-range WBH causes lymphocyte redistribution in
lymphoid tissues
Direct exposure of BALB/c mice to fever-range WBH (core body
temperature, 39.8 ± 0.2°C) for 6 hours dramatically altered the distribution of lymphocytes in various lymphoid tissues (Figure 1). A significant decrease was observed
in the total number of circulating lymphocytes, as reported
previously,16 as well as in the number of
L-selectin-positive and 47 integrin-positive lymphocytes in
peripheral blood (Figure 1). A concomitant increase in lymphocyte
accumulation occurred in PLN (Figure 1) as well as in PP, in which a
2.3-fold increase in the number of lymphocytes was detected (data not
shown; results for PP of WBH-treated mice were significantly different
from those for PP of normothermic mice [P < .02; 3 experiments]). Lymphocyte distribution in the spleen was not altered
by WBH.
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| Figure 1.
Fever-range WBH causes lymphocyte redistribution in
lymphoid tissues.
After WBH treatment of BALB/c mice for 6 hours (core temperature,
39.8 ± 0.2°C), the total number of lymphocytes in lymphoid organs
(peripheral blood [PB], PLN, and spleen) was quantified. L-selectin
and 47 integrin expression on tissue lymphocytes was analyzed by
flow cytometry. Values represent the mean ± SE of 3 independent
experiments. Analysis by unpaired 2-tailed Student t test
showed that the difference between the number of lymphocytes in PB and
PLN of normothermic controls (37.1 ± 0.2°C) and WBH-treated mice
was significant (* indicates P < .02 and **,
P < .03).
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WBH stimulates L-selectin and 47 integrin-dependent adhesion
in splenic lymphocytes
To investigate the mechanisms underlying the increase in
lymphocyte accumulation in PLN and PP, we first examined the effect of
WBH on the adhesion properties of murine lymphocytes (Figure 2). Splenic lymphocytes were isolated
from BALB/c mice treated for 6 hours with fever-range WBH
(39.8 ± 0.2°C), and adhesion of those lymphocytes to PNAd or
MAdCAM-1 on postcapillary HEV of frozen lymphoid tissues was assessed
under mechanical shear conditions. Fever-range WBH significantly
increased lymphocyte adhesion to PLN and PP HEV in comparison to
adhesion of lymphocytes from normothermic mice (36.8 ± 0.2°C;
P < .0001; Figure 2). Hyperthermia-induced lymphocyte
adhesion to PLN HEV depended on both L-selectin and PNAd, as indicated
by inhibition using function-blocking mAbs (ie, Mel-1421
and MECA-79,6 respectively). The increase in lymphocyte
adhesion to PP HEV observed in response to WBH was similarly shown to
depend on 47 integrin and MAdCAM-1, on the basis of mAb blockade
by the 47 integrin-specific mAb DATK3214 and the
MAdCAM-1-specific mAb MECA-367.7 However, the surface density of both L-selectin and 47 integrin on splenocytes was not
altered by WBH, as determined by flow cytometric analysis (data not
shown). The finding that WBH regulated lymphocyte adhesion in vivo
supports and extends recent observations indicating that hyperthermia
acts on lymphocytes in vitro to enhance L-selectin-mediated and
47 integrin-mediated adhesion to HEV without affecting
expression of these homing receptors.9-11
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| Figure 2.
Fever-range WBH stimulates lymphocyte adhesion to PNAd
and MAdCAM-1 on HEV.
Splenic lymphocytes were isolated from BALB/c mice treated for 6 hours
with WBH and from normothermic mice. The cells were then tested for
adhesion to PLN and PP HEV (tissues from non-heat-treated mice) in a
frozen-section adhesion assay under mechanical shear conditions. The
specificity of lymphocyte-HEV adhesion was determined using the
indicated function-blocking mAbs. Values represent the mean ± SE
of 3 independent experiments. The differences between adhesion of
splenocytes from normothermic control mice and WBH-treated mice were
significant (P < .0001 [*] by unpaired 2-tailed Student
t test).
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Fever-range WBH enhances PNAd and MAdCAM-1-dependent adhesion
in HEV
To determine whether fever-like temperature also regulates
adhesiveness of HEV, adhesion assays were performed using frozen lymphoid tissues from BALB/c mice treated for 6 hours with WBH. A
significant increase in the number of lymphocytes bound to HEV was
detected when PBL (human PBL maintained at 37°C) were overlaid on PLN
sections from WBH-treated mice (P < .0008; Figure
3A and 3B). Hyperthermia-induced adhesion
was blocked by antihuman L-selectin-specific DREG-56 mAb22
and MECA-79 mAb.6 The WBH effects were remarkably stable
considering that elevated HEV adhesion was maintained during cryopreservation of PLN tissues and in assays performed at 4°C. The
kinetics of the WBH response were tightly regulated. A moderate increase in HEV adhesion was detected 2 hours after WBH, whereas adhesion was augmented markedly after 6 hours of WBH (Figure 3B). Moreover, HEV adhesion in PLN returned to normal levels within 12 hours
after cessation of WBH (Figure 3C). Similar increases in PLN HEV
adhesion were observed in response to WBH in other murine strains
(C57BL/6 and C3H) and in B-cell-deficient and T-cell-deficient severe
combined immunodeficiency disease mice (S.S.E. et al,
unpublished data, 2000).
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| Figure 3.
Fever-range WBH augments the ability of PLN HEV to
support L-selectin-dependent lymphocyte adhesion.
(A) PLN from BALB/c mice treated for 6 hours with WBH (core
temperature, 39.8 ± 0.2°C) and normothermic controls
(36.9 ± 0.2°C) were used in frozen-section adhesion assays. Arrows
indicate HEV structures containing darkly stained adherent human PBL
indicator cells; bar indicates 50 µm. (B) Quantification of human PBL
adhesion to PLN HEV from normothermic mice and from mice treated for 2 or 6 hours with WBH. Before assay, PBL or PLN tissue sections were
incubated for 30 minutes with the indicated function-inhibiting mAb. *
indicates P < .0008; **, P < .02. (C)
Treatment groups included normothermic controls, mice treated for 6 hours with WBH, and mice treated for 6 hours with WBH and then
maintained at room temperature for an additional 12 hours to allow core
temperatures to return to normal (37.0 ± 0.2°C). PLN were removed
and used in frozen-section adhesion assays to evaluate human PBL
adhesion to HEV. * indicates P < .002. (D) Adhesion of
murine 300.19/L-selectin transfectants and 300.19/L-cyto cells was
evaluated in frozen sections of LN HEV from normothermic mice and mice
treated with WBH for 6 hours. * indicates P < .0007. The
differences between HEV adhesion in PLN from normothermic mice and
WBH-treated mice were significant by unpaired 2-tailed Student t
test.
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To confirm the L-selectin dependence of the WBH response in PLN HEV, we
examined the effect of WBH on the ability of PLN HEV to support
adhesion of murine 300.19 B-cell transfectants that express full-length
human L-selectin (300.19/L-selectin) or 300.19/L-cyto cells that
express a truncated, nonfunctional form of L-selectin lacking the 11 carboxy-terminal amino acids of the cytoplasmic tail.10,13,23 L-selectin-dependent adhesion of
300.19/L-selectin cells was significantly stimulated by WBH
(P < .007), whereas WBH failed to enhance binding of
300.19/L-cyto cells to PLN HEV (Figure 3D). Moreover, because 300.19 transfectants are LFA-1-negative,23 this experiment
excluded any contributions of LFA-1 to hyperthermia-induced adhesion to HEV.
Febrile responses in mice range from 37.5 to 39.8°C after natural
infections (eg, influenza and cecal infection) or stimulation by
inflammatory agents (LPS and turpentine).1,2,8,20 To determine whether increased HEV adhesion occurs in a similar
temperature range, mice were treated with various temperatures of WBH,
and lymphocyte adhesion to lymph node HEV was examined in frozen
sections (Figure 4). Significant
increases in lymphocyte-HEV adhesion were observed when body
temperatures were raised to 38 ± 0.3°C, 39 ± 0.4°C, and
40 ± 0.2°C. To examine the relationship between adhesion and
fever-inducing inflammatory responses, the effects on
L-selectin-dependent binding of lymphocytes to lymph node HEV were
assessed in 2 well-characterized models of fever induction during
sterile inflammatory responses.8,19,20 As shown in Figure
4, a marked increase in L-selectin-dependent adhesion of lymphocytes
to lymph node HEV was detected after fever induction in response to
bacterial LPS, which induces systemic inflammation, or turpentine,
which induces local inflamed abscesses.
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| Figure 4.
Increased PLN HEV adhesion is associated with febrile
temperatures after WBH treatment or inflammation induced by turpentine
or LPS.
The core temperature of mice was raised to the indicated temperature by
either WBH treatment for 6 hours (left panel), injection of turpentine
oil ([Turp], 100 µL given subcutaneously; middle panel), or
injection of LPS (10 µg/kg given intraperitoneally; right panel).
Control mice were given injections of sterile saline. PLN tissues were
isolated at the indicated times, and adhesion of human PBL to HEV of
frozen sections was quantified under shear conditions. Before assay,
selected PBL samples were pretreated with DREG-56 blocking mAb. Data
are the mean ± SD results with triplicate samples; results are
representative of 3 independent experiments. The differences between
HEV adhesion in PLN from normothermic mice and those from mice at
febrile temperatures were significant by unpaired 2-tailed
Student t test. * indicates P < .0001;  , untreated; and  , DREG-56 mAb treated.
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Analysis of the WBH effects on PP HEV adhesion in vivo revealed that
fever-range temperatures stimulated MAdCAM-1 interactions with both
L-selectin and 47 integrin (Figure
5A), representing 2 independent classes
of adhesion molecules.3,4 In this regard, WBH treatment of
mice increased the ability of PP HEV to support MECA-367-sensitive
adhesion of L-selectin-positive and 47 integrin-positive human PBL and of TK1 murine T-cell lymphoma cells, which express 47 integrin but not functional levels of
L-selectin.11,15 WBH also enhanced MAdCAM-1-dependent
adhesion of L-selectin-positive 300.19 transfectant cells (Figure 5A),
which do not express DATK32 mAb-reactive 47 integrin. The
increase in adhesion observed in PP and PLN HEV in response to WBH was
not associated with any apparent changes in HEV expression of MAdCAM-1
or PNAd, as determined by immunofluorescence analysis of frozen-tissue
sections (S.S.E. et al, unpublished data, 2000).
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| Figure 5.
Fever-range temperature stimulates
MAdCAM-1-dependent adhesion in PP HEV during WBH treatment in vivo and
in organ cultures in vitro.
(A,B) PP or pancreatic tissues were isolated from normothermal
controls (core temperature of controls [C] was 36.8 ± 0.2°C) or
mice treated 6 hours with WBH (39.7 ± 0.2°C). Tissues were
cryopreserved and used in adhesion assays. (A) Selected tissue
cryosections were blocked with the MAdCAM-1-specific mAb MECA-367. (B)
Cells were either untreated or treated with DATK32 mAb or DREG-56 mAb.
(C) Bilateral pairs of PLN were isolated from normothermic BALB/c mice,
separated into 2 groups, and cultured for 6 hours in 1 mL complete
medium at 37°C or 40°C in a 5% carbon dioxide incubator. PP
collected from normothermic mouse pairs were cultured at 37°C and
40°C. Lymphoid tissues were then frozen and used in adhesion assays
under shear conditions. To evaluate L-selectin-dependent adhesion of
human PBL to PLN HEV, assays were performed without antibody or in the
presence of function-blocking mAb (DREG-56 or MECA-79). To quantify
47 integrin/MAdCAM-1-dependent adhesion in PP organ cultures,
adherence of TK1 indicator cells to HEV was evaluated without antibody
or in the presence of DATK32 mAb or MECA-367 mAb. Data represent the
mean ± SE values from 3 independent experiments. * indicates
significant differences for the increase in HEV adhesion detected in
response to hyperthermia (P < .0001 by unpaired 2-tailed
Student t test).
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In sharp contrast to the marked effects of WBH on adhesion in
differentiated HEV of PLN and PP (Figure 3 and Figure 5A), WBH failed
to increase adhesive interactions involving either 47 integrin
(assessed by using normothermic TK1 cells) or L-selectin (indicated by
results in normothermic 300.19/L-selectin cells) in squamous,
nonspecialized blood vessels of extralymphoid tissues such as the
pancreas (Figure 5B). To determine whether the lymphoid tissue
microenvironment was sufficient to support HEV responses to fever-like
temperature, PLN and PP organ cultures were treated in vitro with
hyperthermia (40°C for 6 hours) before quantification of HEV adhesion
(Figure 5C). Hyperthermia stimulated L-selectin/PNAd adhesion and
47 integrin/MAdCAM-1 adhesion, respectively, in HEV in PLN and PP
organ cultures (P < .0001), closely paralleling the
responses of these tissues during WBH (Figure 3 and Figure 5A). These
data show that HEV adhesion is regulated in the local lymphoid
microenvironment and does not universally require involvement of other organ systems, including the highly integrated
hypothalamus-pituitary-adrenal axis, which is known to contribute to
physiologic febrile responses.1,2
Effect of WBH on L-selectin and 47 integrin-dependent
lymphocyte homing
Short-term migration studies were performed to establish that
fever-range temperature controls delivery of lymphocytes to LN and PP
through L-selectin and/or 47 integrin-dependent mechanisms. Thus, 300.19/L-selectin cells and TK1 cells were used in these in vivo
homing studies to segregate contributions of L-selectin and 47
integrin in response to hyperthermia. Moreover, the absence of LFA-1 on
300.19/L-selectin cells23 allowed analysis of hyperthermia effects on L-selectin-mediated migration to organized
lymphoid tissues without the participation of LFA-1. The distribution
of adoptively transferred 300.19/L-selectin cells in PLN and PP tissues of normothermic mice was similar to that reported for lymphocytes of
LFA-1-deficient mice.24 Therefore, 1 hour after
injection, most 300.19/L-selectin cells were localized in the lumen of
PLN and PP HEV, whereas extravasation into tissue parenchyma was more limited (ie, the proportion of 300.19/L-selectin cells within the lumen
of PLN/PP HEV compared with cells within tissue parenchyma was
77% ± 1% and 23% ± 1%, respectively, with a total of
84 ± 5 cells/mm2 detected in PLN or PP tissues).
In the first series of experiments that examined hyperthermia effects
on L-selectin-mediated migration, PKH-26-labeled 300.19/L-selectin cells and FITC-labeled 300.19/L-cyto cells were simultaneously injected intravenously into BALB/c mice that were pretreated with WBH
for 6 hours. After 1 hour, lymphoid tissues were removed to quantify
the relative accumulation of fluorescence-labeled cells in WBH-treated
mice compared with migration of the same cell types in normothermic
control mice (Figure 6). WBH caused a
greater than 4-fold increase in localization of 300.19/L-selectin cells in PLN relative to results in normothermic controls. WBH also stimulated accumulation of 300.19/L-selectin cells in PP and mesenteric LN (MLN), although to a lesser extent than in PLN, possibly reflecting the requirement for 47 integrin for efficient homing to these tissues.3,4,25 In sharp contrast, WBH failed to augment localization of 300.19/L-cyto cells in PLN, MLN, or PP. The
differences between localization of 300.19/L-selectin cells and
300.19/L-cyto cells in PLN, MLN, and PP in WBH-treated mice were
significant (P < .0001), supporting the conclusion that
WBH stimulates L-selectin-dependent accumulation of cells in lymphoid
tissues.
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| Figure 6.
Fever-range hyperthermia stimulates
L-selectin-dependent and 47 integrin-dependent migration of
lymphocytes to LN and PP.
The 300.19/L-selectin cells and TK1 cells were cultured at 37°C
or treated for 6 hours in vitro with fever-like hyperthermia (Cell/HT;
40°C), and then labeled with PKH-26 fluorescent dye. PKH-26-labeled
cells were mixed with equivalent numbers of FITC-labeled internal
standard cells (300.19/L-cyto cells or TK1 cells treated with DATK32
mAb) and injected intravenously into normothermic control mice (core
temperature, 36.8 ± 0.2°C) or mice pretreated with WBH
(39.7 ± 0.2°C). After 1 hour, the number of fluorescent cells in
single-cell suspensions of lymphoid tissues was determined. Data
represent the relative accumulation of cells in lymphoid tissues,
expressed as a percentage of control cells (ie, cells of the same type
maintained at 37°C) that were labeled identically and injected into
normothermic mice. Therefore, 100% in the graph (broken lines)
represents the behavior of cells cultured at 37°C after injection
into normothermic control mice. Values are the mean ± SE of 3 independent experiments. The difference in accumulation of
300.19/L-selectin cells or TK1 cells relative to internal standard
cells (300.19/L-cyto cells or DATK32-treated TK1 cells,
respectively) was significant by unpaired 2-tailed Student t
test (* indicates P < .0001; and **,
P < .005). The SEM for 300.19/L-cyto cells and
DATK32-treated TK1 cells was less than or equal to 10% (not shown).
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Hyperthermia pretreatment of 300.19/L-selectin cells in vitro before
injection into normothermic mice also markedly increased the level of
accumulation in PLN, MLN, and PP compared directly with homing of
300.19/L-selectin cells maintained at 37°C (P < .0001;
Figure 6). However, combined hyperthermia treatment of cells and
WBH-treatment of mice did not result in additive or synergistic effects
on L-selectin-dependent accumulation in secondary lymphoid tissues.
These data suggest that hyperthermia responses in lymphocytes and HEV
functionally converge on a common adhesion mechanism. A significant
increase in 300.19/L-selectin cell migration to PLN and PP (3-fold and
1.75-fold, respectively) was also observed after only 2 hours of
stimulation by WBH or in vitro hyperthermia treatment of
300.19/L-selectin cells (data not shown).
Similar regulation of 47 integrin-dependent homing of TK1 cells
to PP and MLN was observed in response to WBH or direct hyperthermia
treatment of TK1 cells (Figure 6). Specifically, 6 hours of WBH
treatment of mice or hyperthermia pretreatment of TK1 cells
independently resulted in a greater than 3-fold increase in homing to
PP and MLN. Hyperthermia did not augment TK1 cell homing to PLN,
consistent with previous findings indicating that 47 integrin
does not normally participate in lymphocyte homing to
PLN.3,24,25 The increase in TK1 homing to PP and MLN was significant when compared directly with localization of DATK32 mAb-treated TK1 cells (P < .005), confirming the role of
47 integrin in directing lymphocyte homing to lymphoid tissues in response to hyperthermia. Again, combined WBH and hyperthermia treatment of TK1 cells in vitro did not further augment 47
integrin-dependent migration to PP or MLN. In agreement with evidence
that L-selectin and 47 integrin do not contribute to lymphocyte
localization in spleen,3,25 neither WBH nor hyperthermia
treatment in vitro influenced the distribution of 300.19/L-selectin
cells or TK1 cells in this tissue (Figure 6). Furthermore, fever-like
hyperthermia did not alter the proportion of labeled cells in
peripheral blood, despite increased migration of fluorescent cells to
LN and PP. These observations likely reflect the relatively small
percentage (0.5%-2%) of total cells injected intravenously that
traffic to LN and PP during 1-hour short-term homing
studies25 (S.S.E. et al, unpublished data, 1996).
| |
Discussion |
This study provides the first evidence of an active role for
fever-like temperatures in amplifying lymphocyte delivery to secondary
lymphoid tissues in vivo through the regulation of lymphocyte adhesion
to HEV. Unexpectedly, fever-like hyperthermia affected adhesion in 2 distinct cellular targets. Hyperthermia stimulated lymphocyte adhesion
mediated by L-selectin and 47 integrin, as well as HEV adhesion
dependent on PNAd and MAdCAM-1. Notably, hyperthermia regulation of
lymphocyte-HEV interactions, like the endogenous fever
response,1,2 is evolutionarily conserved in species that
diverged more than 180 million years ago (ie, mouse and
human).26 The 3- to 4-fold increase in lymphocyte migration to LN and PP that occurred in response to hyperthermia potentially represents a profound increase in the opportunity for naive
lymphocytes to encounter foreign pathogens during immune surveillance
of secondary lymphoid tissues. Emerging evidence that subsets of memory
cells share recirculation routes of naive lymphocytes through common
expression of adhesion molecules and chemokine receptors (ie,
L-selectin, 47 integrin, and the CCR7 chemokine
receptor3,4,27) also raises the possibility that the
thermal element of fever promotes delivery of both naive lymphocytes and antigen-primed memory subsets to LN and PP.
Hyperthermia exerts tight control on lymphocyte-endothelial adhesion. A
marked increase in L-selectin-dependent and 47
integrin-dependent lymphocyte-endothelial adhesion and homing to LN
and PP was detected within 2 to 6 hours of stimulation by hyperthermia.
Importantly, lymphocyte delivery to HEV in response to fever-range
temperatures temporally precedes the 8- to 24-hour interval during
which antigen-specific T cells initially become activated in secondary
lymphoid tissues during a primary immune response.28 The
effects of hyperthermia on adhesion are also tightly regulated with
respect to the endothelial target. Robust increases in adhesion were
observed in cuboidal, differentiated HEV of LN and PP but not in
squamous, less-differentiated endothelium of nonlymphoid tissues (ie,
pancreas). Moreover, the specificity of HEV adhesion was not
compromised during the hyperthermia response. Thus, WBH selectively
amplified L-selectin-dependent but not 47 integrin-dependent
delivery of cells to PLN HEV. Preliminary studies have suggested that
WBH also increases adhesion of HEV-like vessels in inflamed
lesions.29 Selective regulation of adhesion in
differentiated HEV but not squamous endothelium by fever temperatures
would serve to focus the immune response to lymphoid tissues and sites
of infection while preventing an unproductive exodus of lymphocytes to
other tissues during a physiologic febrile episode.
Evidence that in vitro hyperthermia treatment stimulates
L-selectin-dependent and 47 integrin-dependent adhesion in
lymphocytes and in HEV of lymphoid tissue organ cultures indicates that
the hypothalamus-pituitary-adrenal axis is not pivotal for this
response. Although the molecular mechanisms underlying
hyperthermia-induced adhesion are not known, it is probable that local
cytokine networks are involved. In this regard, hyperthermia promotes
the release or bioactivity of several proinflammatory cytokines,
including IFN-, tumor necrosis factor (TNF-), interleukin
(IL) 6, and IL-1.1,2,19 These cytokines were found to
enhance L-selectin and 47 integrin adhesion in
vitro12 (S.S.E. et al, manuscript in preparation),
directly paralleling the hyperthermia effects. Moreover, fever
induction in response to LPS or turpentine is associated with high
systemic levels of IL-6, TNF-, and IL-1,1,2,8,19,20 which could be mechanistically related to the marked increase in
lymphocyte-HEV adhesion observed in the current study. Fever-range temperatures stimulate L-selectin and 47 integrin adhesion in lymphocytes through the release of soluble autocrine factors, without
increasing the surface density of these molecules (reported here and
previously9-12). These findings suggest that hyperthermia enhances the affinity or avidity of selected lymphocyte adhesion molecules, possibly by altering interactions with the nonionic detergent-insoluble cytoskeletal matrix10 or the overall
organization of the spectrin-actin-based cytoskeleton, as observed
previously in response to fever-range
hyperthermia.17,18,30
It remains to be determined whether increases in HEV adhesion reflect
direct effects of hyperthermia on endothelial cells or indirect effects
on stromal cells (eg, monocytes, fibroblasts, and dendritic cells) in
the microenvironment of organized secondary lymphoid tissues. Studies
are in progress to determine whether the remarkably stable changes in
lymphocyte-HEV adhesion observed here can be explained by biochemical
modifications (eg, altered sulfation or sialylation of PNAd or
MAdCAM-1), conformational changes, increased clustering, or changes in
the lateral mobility of adhesion molecules in lymphocyte or endothelial
plasma membranes.3-5 Collectively, our findings provide
new evidence for a unifying mechanism by which febrile temperatures
dynamically modulate regional recruitment of circulating lymphocytes to
tissues during physiologic responses to infection and inflammation.
| |
Acknowledgments |
We thank E. C. Butcher and G. S. Kansas for sharing the
TK1 and 300.19 L-selectin cell lines, respectively; C. C. Stewart for advice on flow cytometry; M. McGarry and J. Lau for assistance in
turpentine inflammatory studies; R. A. Bruce, C.-Y. Tsao, and A. Sumlin for technical support; and J. D. Black for critical review
of the manuscript.
| |
Footnotes |
Submitted October 26, 2000; accepted December 23, 2000.
Supported by grants from the National Institutes of Health (CA79765
[S.S.E.], CA71599 [E.A.R.], and P30 CA16056-21), the Department of
Defense (DAMD17-8-8311 [S.S.E.]), the Roswell Park Alliance Foundation (S.S.E. and E.A.R.), and the Cancer Research Institute (J.R.O.).
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
Reprints: Sharon S. Evans, Department of Immunology, Roswell
Park Cancer Institute, Carlton and Elm Streets, Buffalo, NY 14263.
| |
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Abstract
Introduction