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The Journal of Immunology, 1998, 160: 961-969.
Copyright © 1998 by The American Association of Immunologists

Fever-Range Hyperthermia Enhances L-Selectin-Dependent Adhesion of Lymphocytes to Vascular Endothelium1

Wan-Chao Wang*, Lorin M. Goldman*, David M. Schleider*, Michelle M. Appenheimer*, John R. Subjeck{ddagger}, Elizabeth A. Repasky{dagger} and Sharon S. Evans2,*,{dagger}

Departments of * Molecular Medicine, {dagger} Molecular Immunology, and {ddagger} Molecular and Cellular Biology, Roswell Park Cancer Institute, Buffalo, NY 14263


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The L-selectin leukocyte adhesion molecule plays an important role in controlling leukocyte extravasation in peripheral lymph nodes and at sites of tissue injury or infection. Although febrile responses during infection and inflammation are associated with enhanced immune activity, the contribution of fever-range temperatures to controlling lymphocyte recruitment to tissues has not been previously examined. In this report we provide evidence that direct exposure of lymphocytes to fever-range temperatures (38–41°C) in vitro for 9 to 24 h resulted in a >100% increase in L-selectin-dependent adhesion of these cells to lymph node high endothelial venules (HEV). Moreover, culture of lymphocytes under hyperthermia conditions markedly enhanced the ability of these cells to traffic in an L-selectin-dependent manner to peripheral lymph nodes, mesenteric lymph nodes, and Peyer’s patches. In contrast, febrile temperatures did not increase LFA-1 function as assessed by measuring lymphocyte adhesion to ICAM-1–3T3 transfectants. Fever-range hyperthermia further did not increase L-selectin surface density on lymphocytes or L-selectin-dependent recognition of soluble carbohydrate substrates; however, a marked increase in ultrastructural immunogold-labeling of L-selectin was observed in response to thermal stimuli. These results suggest that elevated temperatures enhance L-selectin adhesion and/or avidity through the regulation of L-selectin conformation or organization in the plasma membrane. Finally, the observed thermal effects on L-selectin adhesion were attributed to soluble factors in the conditioned medium of heat-treated cells. Taken together, these data provide new insight into the potential physiologic role of the febrile response in enhancing lymphocyte recruitment to tissues through the regulation of L-selectin adhesion.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
An active immune response is dependent on the continuous recirculation of lymphocytes through peripheral lymphoid tissues such as lymph nodes and Peyer’s patches (PP).3 Immune effector cells are also actively recruited to sites of local injury, infection, or inflammation. Lymphocyte extravasation involves multiple adhesion molecules that mediate lymphocyte binding to endothelial cells lining vascular beds. The L-selectin leukocyte adhesion molecule initiates the attachment and tethering of lymphocytes to peripheral lymph node addressins (PLNAd) on the luminal surface of specialized high endothelial venules (HEV) in lymph nodes and PP (1, 2). L-selectin is specifically adapted to mediate the initial attachment of lymphocytes to HEV under physiologic shear forces within vessels. Firm adhesion and extravasation of lymphocytes across HEV are dependent on the interaction of leukocyte LFA-1 with ICAM-1/2 on endothelial cells (3). The {alpha}4ß1 and {alpha}4ß7 integrins have also been implicated in the recruitment of lymphocytes to inflamed tissues through binding to the endothelial cell ligands, VCAM-1 and mucosal addressin cell adhesion molecule-1 (MAdCAM-1) (1, 3). Local release of proinflammatory cytokines such as IL-1ß, TNF-{alpha}, and IFN-{gamma} at sites of infection or inflammation is associated with the induced expression of adhesion molecules (i.e., PLNAd, ICAM-1, VCAM-1, MAdCAM-1, and E-selectin) on endothelial surface membranes, thereby stimulating leukocyte emigration into tissues (1, 3, 4, 5).

Local increases in temperature at sites of inflammation and systemic fever are cardinal features of host responses to pathogenic stimuli. Proinflammatory cytokines such as IL-1ß and TNF-{alpha} are pyrogenic and have been implicated in the generation of endogenous fever, an ancient, highly conserved response to infection (6, 7, 8, 9, 10). Febrile-range temperatures are associated with enhancement of the immune response through augmentation of a) T cell proliferation and cytotoxicity (10, 11, 12), b) the antiviral activity of IFNs (10, 13, 14), and c) the neutralizing capacity of Abs (6, 15). Fever may also have deleterious effects on immune defenses under certain circumstances. Even moderately elevated temperatures (e.g., 1°C above physiologically normal temperatures) suppress NK cell function (10, 16) while high temperatures inhibit CTL responses (17). It is generally assumed that fever-range temperatures positively influence leukocyte recruitment to tissues through changes in blood flow hemodynamics due to vasodilation. However, several studies have suggested that fever-range hyperthermia plays a more active role in directing cell migration into tissues. Specifically, fever temperatures have been shown to increase a) neutrophil migration, motility, and chemotaxis (10, 18, 19), b) platelet adhesion (20), and c) ICAM-1 expression on vascular endothelium (21). In addition, we have previously demonstrated that febrile-range hyperthermia treatment of lymphocytes alters the intracellular organization or expression of cytoskeletal proteins (i.e., spectrin), heat shock proteins (hsp70 family), and protein kinase C (22, 23), events that may be linked to lymphocyte activation and adhesion. The effect of physiologic fever-range temperatures on the expression or function of adhesion molecules on lymphocytes has not been previously examined.

The present study was undertaken to investigate whether direct exposure of lymphocytes in vitro to fever-range temperatures influences the adhesion potential of these cells. Data presented in this study demonstrate that elevated temperatures markedly enhance L-selectin-mediated adhesion of human lymphocytes to lymph node HEV in vitro in a time- and temperature-dependent manner. Moreover, direct exposure of lymphocytes to fever-range temperatures in vitro enhances their ability to traffic to PLN, MLN, and PP in vivo. In contrast, LFA-1 function was not augmented by hyperthermia treatment of lymphocytes. These results demonstrate a previously unrecognized mechanism by which febrile responses may act in vivo to enhance immune surveillance of lymphoid tissues and augment lymphocyte recruitment to infected or inflamed tissues. In addition, these studies provide insight into the potential mechanisms operative during hyperthermia therapy in cancer patients.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell isolation and culture

PBMC were isolated from normal donor buffy coat leukocyte concentrates (American Red Cross, Buffalo, NY ) by Ficoll/Hypaque centrifugation, as described previously (24, 25, 26). Following removal of adherent cells, the PBL population was cultured at a final concentration of 4 x 106 cells/ml in RPMI 1640 medium (Life Technologies, Grand Island, NY) containing 10% FCS (Life Technologies), 2 mM L-glutamine, 100 U/ml penicillin, and 50 µg/ml streptomycin. Human PBL were cultured at temperatures ranging from 34.5°C to 41°C in a 5% CO2 incubator.

The ICAM-1–3T3 transfectants and 3T3-Neo controls have been described previously by Berman et al. (27) and were generously provided by Dr. W. Muller (Rockefeller University, New York, NY). These cells were maintained in culture in DMEM (Life Technologies) with 10% FCS, 1 mg/ml G418 (Life Technologies), and 0.4 mg/ml hygromycin (Calbiochem, La Jolla, CA). The ICAM-1-transfectant population was >95% positive for ICAM-1. 3T3-Neo controls were >99% ICAM-1 negative.

Ab and reagents

The following mouse IgG mAb specific for human L-selectin were used: DREG-56 (28) (unconjugated mAb was kindly provided by Dr. E. Butcher, Stanford University, Stanford, CA; FITC-conjugated mAb was obtained from Immunotech, Inc. Westbrook, ME), Leu-8-FITC (Becton Dickinson, Sunnyvale, CA), and TQ1 (Coulter Immunology, Hialeah, FL). The MEL-14 mAb (29), a rat IgG2a specific for mouse L-selectin, was obtained from the American Type Culture Collection (ATCC, Rockville MD). Additional mAb specific for the following adhesion molecules were also used: human LFA-1 (unconjugated TS1/22 mAb from ATCC; FITC-conjugated mAb from Endogen, Inc., Boston, MA), {alpha}4 integrin (Telios Pharmaceuticals, Inc., San Diego, CA), and PLNAd (MECA-79, a rat IgM, generous gift of Dr. E. Butcher). Control murine mAb A4 (IgG1) was kindly provided by Dr. R. Ward (Roswell Park Cancer Institute, Buffalo, NY). Goat anti-mouse IgG bound to 20 nm colloidal gold was purchased from Goldmark Biologics (Phillipsburg, NJ). PMA and fibronectin were purchased from Sigma Chemical Co. (St. Louis, MO).

Flow cytometric analysis

Direct immunofluorescence analysis of the relative expression of lymphocyte cell surface molecules was performed using a FACScan (Becton Dickinson) in the Roswell Park Flow Cytometry facility as described previously (24, 25, 26). A total of 106 cells were washed with PBS/0.02% sodium azide and then incubated with 0.1 mg/ml mouse Ig (Sigma Chemical Co.) for 10 min at 4°C to block Fc receptor sites. Cells were then incubated with saturating amounts of fluorochrome-labeled mAb or isotype-matched control mAb for 30 min at 4°C, washed in PBS/sodium azide, and fixed in 1% formaldehyde/PBS. A total of 10,000 events were collected, and analysis was performed using Winlist 1.0 (Verity Software House, Inc., Topsham, ME).

PPME binding assay

Analysis of L-selectin-dependent binding of lymphocytes to PPME, the phosphomonoester core from Hansenula hostii phosphomannan, was performed as described (24, 25, 30). PBL at a concentration of 5 x 106 cells/ml were incubated for 15 min at 4°C either in medium alone or with the L-selectin-specific mAb TQ1 (10 µg/ml). Without washing, cells were then incubated with a 1:200 dilution of fluorescein-conjugated PPME (generous gift of Dr. L. Stoolman, University of Michigan, Ann Arbor, MI) for 30 min at 4°C and then analyzed immediately by flow cytometry. Fluorescence measurements of FITC-conjugated PPME binding were made with logarithmic amplification.

Ultrastructural immunolocalization of L-selectin

Localization of L-selectin on human PBL was determined by immunogold TEM. Following incubation with 10 µl of goat serum (diluted 1:2) to block potential Fc receptor sites, PBL were labeled on ice with primary mAb (30 µg/ml of DREG-56 or isotype-matched negative control Ab), stained with colloidal gold-conjugated secondary mAb, fixed in 3% glutaraldehyde, and processed for TEM by the Roswell Park Cell Analysis Facility. Sections were viewed in an Elmiskop 101 electron microsope (Siemens Corp., Iselin, NJ). Blind analysis of triplicate specimens was performed by counting gold particles associated with distinct surface structures (microvilli or cell body) on a total of 100 cells in each specimen.

Stamper-Woodruff frozen section assay

Lymphocyte binding to HEV was assessed as described (25, 28, 31). Lymphocytes were resuspended at 5 x 107 cells/ml in complete medium and then incubated for 30 min at room temperature with or without saturating amounts of L-selectin-specific blocking mAb (DREG-56 or MEL-14, which recognizes human or murine L-selectin, respectively). Lymphocytes (5 x 106 cells in 100 µl) were then overlaid onto 12-µm-thick cryosections of BALB/c lymph nodes mounted on glass slides. Previous studies have established that L-selectin-binding specificity is maintained during assay of human lymphocyte adhesion to mouse lymph node HEV (28). In some instances, lymph node sections were preincubated 30 min at 4°C with MECA-79 mAb or isotype-matched negative control Ab. Slides were rotated at 112 rpm (Labline Instrument, Inc., Melrose Park, IL) at 4°C for 30 min, and nonadherent cells were removed by gentle washing in cold PBS. Slides were fixed vertically in 3% glutaraldehyde/PBS for 1 h, permeabilized in 70% ethanol, and stained with 0.5% toluidine/absolute ethanol. A total of 300 to 500 HEV were examined by light microscopy, and data are expressed as the mean number of lymphocytes bound per HEV +/- SD; each sample was assayed in triplicate. Exogenously added lymphocytes exhibited a darkly stained, round appearance and were distinguished from histologically distinct tissue lymphocytes and HEV.

LFA-1-dependent cell adhesion assay

LFA-1-dependent adhesion of human PBL to human ICAM-1-transfected 3T3 fibroblasts was evaluated essentially as described by Berman et al. (27). Following incubation of PBL in the absence or presence of saturating concentrations of anti-LFA-1 mAb (TS1/22) for 30 min on ice, 2 x 105 cells (100 µl) were then added to confluent monolayers of ICAM-1–3T3 transfectants or Neo-transfectants grown on fibronectin-coated 96-well plates. Each experimental condition was set up in six replicate wells. Following incubation for 30 min at 37°C, wells were washed and then fixed in 3% glutaraldehyde (100 µl/well). The number of adherent lymphocytes bound to transfectant 3T3 cells was counted in three to four random high power fields per well, results were averaged, and data are expressed as the mean +/- SD of six replicate samples.

In vivo trafficking assay

Spleen cell suspensions from 6- to 8-wk-old BALB/c mice were prepared in RPMI 1640 medium, and erythrocytes were lysed with a 0.83% ammonium chloride solution. Cells were then cultured for 12 h at 37°C or 40°C. FITC labeling of hyperthermia-treated cells was performed as described (29, 32, 33). Briefly, 5 x 107 cells were incubated at 37°C for 20 min with 1 ml of PBS containing 3% FCS and 100 µl of a 300 µg/ml solution of FITC (Sigma Chemical Co.). PKH26 labeling of lymphocytes maintained at 37°C was performed as recommended by the manufacturer (Sigma Immunochemicals Co.). Cells (2 x 107) were resuspended in 1 ml of PKH26 diluent, immediately added to an equal volume of a 6 µM PKH26 solution, and then incubated for 3 min at room temperature. After labeling, cells were washed in medium containing FCS and resuspended in PBS. FITC-labeled lymphocytes (40°C treated cells) were mixed with an equal number of an internal standard population of PKH26-labeled lymphocytes (maintained at 37°C) and injected i.v. into the tail vein of syngeneic recipients (5 x 107 total cells per mouse in 100 µl). An aliquot of the initial mixtures (before injection) was analyzed by fluorescence microscopy to calculate the ratio of FITC-PKH26-labeled cells (Ri). After 1 h, single cell suspensions from peripheral blood, spleen, PLN (cervical, axillary, brachial, inguinal, and sciatic), MLN, and PP were prepared and the relative number of FITC- and PKH26-labeled cells was quantified by fluorescence microscopy as described (29, 32). A minimum of 600 fluorescent cells was counted for each replicate sample, and the ratio of FITC-PKH26-labeled cells (Ro) was determined as reported previously (29, 32, 33).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Fever-range hyperthermia enhances L-selectin-dependent adhesion of human PBL to peripheral lymph node HEV

The effects of fever-range temperatures on lymphocyte adhesion to vascular endothelium was assessed in a Stamper-Woodruff adhesion assay. In this assay, human PBL were overlaid onto frozen sections of murine lymph nodes, and adhesion to morphologically distinct postcapillary HEV was assessed under nonstatic conditions to simulate blood flow dynamics in vivo. The data shown in Figure 1GoA indicate that incubation of lymphocytes at 40°C for 24 h results in a marked increase in lymphocyte binding to HEV in vitro. Quantification of lymphocyte binding to HEV demonstrated that hyperthermia treatment increased lymphocyte-HEV interactions by approximately 100% (Fig. 1GoB). Hyperthermia-induced enhancement of lymphocyte binding to HEV was further shown to be L-selectin dependent, as indicated by evidence that adhesion was inhibited greater than 90% by the DREG-56 mAb that blocks L-selectin function as well as by the MECA-79 mAb that binds L-selectin endothelial cell ligands (i.e., PLNAd). Equivalent increases in L-selectin-dependent adhesion were also observed when the assay was performed in the presence of EDTA and excess CaCl2 (data not shown), consistent with the known calcium requirement of the L-selectin adhesion molecule (1, 2, 30). These results indicate that hyperthermia induces adhesion of lymphocytes to blood vessels through a physiologically relevant adhesion pathway and, thus, does not reflect nonspecific cell-to-cell interactions that could potentially be promoted by heat treatment.



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  		FIGURE 1. Fever-range hyperthermia enhances L-selectin-dependent adhesion of human PBL to lymph node HEV in vitro. A, Human PBL were cultured 24 h at 37°C (a) or 40°C (b), and adhesion to cryosections of BALB/c PLN HEV was assayed as described. Darkly stained, exogenous lymphocytes that have adhered to HEV structures are indicated by arrows. B, Following culture of lymphocytes for 24 h at 37°C (open bars) or 40°C (black bars), PBL or lymph node sections were incubated 30 min with the indicated function-inhibiting mAb (50 µg/ml of DREG-56, a murine IgG1, or MECA-79, a rat IgM) or with isotype-matched control mAb, and lymphocyte adhesion was assessed in a Stamper-Woodruff adhesion assay. Error bars denote SD of three replicate samples. Data are representative of >ten independent experiments.

 
Studies were also performed to examine the effect of fever-range hyperthermia on the function of LFA-1, a second adhesion molecule required for lymphocyte migration into tissues. For these studies, human PBL were adhered to monolayers of 3T3 cells transfected with ICAM-1, a ligand for LFA-1 (Fig. 2Go). The level of LFA-1-specific adhesion in this assay was determined using a function-inhibiting mAb against the {alpha}-chain of LFA-1. Identical, low basal levels of LFA-1-specific adhesion of PBL to ICAM-1 transfectants and Neo-transfectants were detected, consistent with previous reports demonstrating that LFA-1 is not constitutively active on circulating lymphocyte populations (3, 34). As expected based on evidence that LFA-1 avidity is increased by PMA activation (3, 34), incubation of PBL with PMA for 24 h triggered a greater than 4-fold increase in LFA-1 dependent adhesion of PBL to ICAM-1 transfectants without affecting adhesion to Neo-transfectant controls. Although incubation of PBL at 40°C for 24 h appeared to slightly increase the level of adhesion to both ICAM-1 transfectants and Neo-transfectants, this adhesion was not LFA-1 dependent. Thus, in sharp contrast to the marked increase in L-selectin-dependent adhesion of PBL to HEV observed in the Stamper-Woodruff adhesion assay, incubation of PBL under hyperthermia conditions failed to significantly increase LFA-1-specific binding to ICAM-1 transfectants.



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  		FIGURE 2. Fever-range temperatures did not affect LFA-1-dependent adhesion of PBL to 3T3-ICAM-1 transfectants. Human PBL were cultured for 24 h at 37°C or 40°C in the absence or presence of 50 ng/ml PMA. Thirty minutes before the assay, lymphocytes were incubated with anti-LFA-1 blocking mAb (TS1/22), as indicated, and then added to 3T3 monolayers grown on fibronectin-coated tissue culture wells. After incubation for 30 min at 37°C, nonadherent cells were removed, and the number of lymphocytes bound per field was quantified. ICAM-1 transfectants supported a slightly elevated basal level of binding of unstimulated PBL compared with control Neo-transfectants; note, however, that the level of LFA-1-specific adhesion of resting PBL to ICAM-1-and Neo-transfectants was similar. Furthermore, although the level of LFA-1-specific adhesion of PBL to ICAM-1 transfectants was increased by PMA, hyperthermia did not significantly augment LFA-1-dependent adhesion of unstimulated or PMA-stimulated cells to ICAM-1 transfectants. Results are representative of three independent experiments.

 
In vivo homing of hyperthermia-treated lymphocytes

To determine the effect of hyperthermia on the migration of lymphoid cells in vivo, murine splenocytes were incubated 12 h at 40°C, labeled with FITC, and injected i.v. into syngeneic mice. FITC-labeled cells were injected together with an equivalent number of PKH26-labeled splenocytes that were maintained at 37°C as an internal standard to assess the normal level of recirculation or homing. Mice were sacrificed 1 h after injection and the relative number of FITC- and PKH26-labeled cells in single cell suspensions of peripheral blood, spleen, PLN, MLN, and PP was determined by fluorescence microscopy. At 1 h, the accumulation of FITC-labeled hyperthermia-treated cells in lymph nodes and PP was significantly greater than PKH26-labeled control cells (Fig. 3Go). In contrast, incubation of murine splenocytes at 40°C did not alter the localization of these cells in the peripheral blood or spleen. Notably, previous studies have established that lymphocyte homing to lymph nodes and PP, but not to spleen, is L-selectin dependent (1, 2, 3, 29, 33). The approximately twofold increase in the accumulation of FITC-labeled cells in lymph nodes and PP observed in short-term homing studies directly paralleled the level of increase in L-selectin-dependent adhesion of hyperthermia-treated murine splenocytes to lymph node HEV detected in vitro in a Stamper-Woodruff assay (data not shown). Moreover, preincubation of both control and heat-treated cells with the MEL-14 mAb, which is specific for mouse L-selectin, markedly suppressed lymphocyte homing to PLN, MLN, and PP in vivo (>70% inhibition, data not shown), further suggesting that the effects of fever-range hyperthermia on lymphocyte trafficking to lymph nodes and PP are L-selectin dependent.



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  		FIGURE 3. Fever-range hyperthermia enhances L-selectin-dependent homing of lymphocytes to lymph nodes and PP. Following incubation at 40°C for 12 h, murine splenocytes were FITC labeled, added to an equal number of an internal standard PKH-26-labeled cells (maintained at 37°C), and injected i.v. into recipient mice. After 1 h, single cell suspensions were obtained from peripheral blood (PB), spleen, PLN, PP, and MLN, and the numbers of fluorescent cells were identified by fluorescence microscopy. The ratio of FITC-PKH-26-labeled cells present in each organ (Ro) was determined and normalized by dividing it with the Ri ratio determined for the injected cell mixture. Results represent the mean ± SD of three independent experiments.

 
Hyperthermia-induced enhancement of L-selectin adhesion requires continuous, limited duration exposure of PBL to fever-range temperatures

To examine the kinetics of induction of L-selectin-dependent adhesion of lymphocytes to HEV, human PBL were cultured at 40°C for various times up to 72 h, and adhesion was examined in the Stamper-Woodruff in vitro assay. The data shown in Figure 4Go indicate that the effects of fever-range hyperthermia on lymphocyte adhesion are tightly regulated in a time-dependent manner. Marked enhancement of L-selectin-dependent lymphocyte adhesion required continuous exposure to hyperthermia conditions over a period of 9 to 24 h, whereas only moderate effects on adhesion were observed following short-term thermal stimulation of lymphocytes for 2 h. Moreover, prolonged exposure of lymphocytes to 40°C for 48 and 72 h was accompanied by a return to normal levels of adhesion. The studies shown in Figure 4Go further support the notion that the effects of febrile temperatures on lymphocyte adhesion are reversible. In this regard, initial incubation of PBL at 40°C for 12 h (i.e., conditions that maximally stimulate lymphocyte-HEV interactions), followed by subsequent culture at 37°C for 12 h, failed to markedly enhance L-selectin-dependent adhesion of lymphocytes to lymph node HEV above control levels.



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  		FIGURE 4. Continuous, limited duration fever-range hyperthermia is required to increase L-selectin-dependent adhesion of lymphocytes to HEV. Human PBL were incubated for 0 to 72 h at 40°C before the analysis of L-selectin adhesion. In some instances, PBL were initially incubated at 40°C for 12 h, followed by subsequent culture at 37°C for 12 h, as indicated. Hyperthermia treatment did not adversely affect lymphocyte viability under these conditions (data not shown). Immediately before assay, lymphocytes were incubated 30 min at room temperature with 50 µg/ml of A4, an isotype-matched negative control mAb (black bars), or DREG-56 (open bars), and adhesion to lymph node HEV was assessed as described in Materials and Methods. Error bars denote SD of triplicate samples. Data are representative of three independent experiments.

 
The effect of various temperatures on lymphocyte adhesion to HEV was examined by incubating PBL for 24 h at temperatures ranging from 34.5°C to 41°C (Fig. 5Go). Incubation of lymphocytes at subnormal temperatures such as 34.5°C or 36°C resulted in a modest inhibition of L-selectin-dependent adhesion. Conversely, incremental increases in L-selectin-dependent lymphocyte adhesion to HEV were observed when cells were cultured at fever-range temperatures ranging from 38°C to 41°C. Interestingly, increased levels of adhesion were observed even at a temperature in the high range of physiologic febrile responses, e.g., 41°C.



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  		FIGURE 5. Temperatures within the physiologic range of febrile responses increase L-selectin-dependent lymphocyte-HEV interactions. Following culture of human PBL for 24 h at the indicated temperatures, cells were incubated 30 min at room temperature with 50 µg/ml of A4, an isotype-matched negative control mAb (closed circles) or DREG-56 (open circles). Lymphocyte adhesion to lymph node HEV was quantified as described. Error bars denote SD of triplicate samples. Results are representative of three independent experiments.

 
Fever-range hyperthermia does not increase L-selectin surface expression or recognition of soluble carbohydrate ligands

L-selectin adhesion can be potentially regulated at multiple levels through the modulation of the cell surface density, affinity, or avidity of this adhesion molecule. To determine whether fever-range temperature enhances L-selectin binding to HEV by up-regulating L-selectin expression, PBL were incubated for 24 h at 40°C, and then the surface density of L-selectin was examined by flow cytometric analysis. The data shown in Figure 6Goa demonstrate that hyperthermia conditions do not alter the cell surface expression levels of L-selectin, as indicated by the immunofluorescence profiles of PBL stained with FITC-conjugated DREG-56 mAb. Identical results were obtained by direct or indirect immunofluorescence analysis using DREG-56 (Fig. 6Goa) or another L-selectin-specific mAb, anti-Leu-8 (data not shown). The conclusion that hyperthermia does not alter L-selectin protein levels in lymphocytes was further confirmed by Western blot analysis (not shown). Thus, the stimulatory effects on adhesion observed in response to hyperthermia cannot be attributed to increased L-selectin density on the surface of PBL. Fever-range temperatures similarly did not affect the surface density of LFA-1 or {alpha}4 ß integrins (Fig. 6Go, b and c).



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  		FIGURE 6. Fever-range temperature does not alter L-selectin cell surface expression or L-selectin-dependent binding of lymphocytes to PPME. Human PBL were cultured for 24 h at 37°C (solid histogram) or 40°C (dotted histogram). Cells were then stained with fluorochrome-labeled mAb specific for the indicated cell surface molecules (a-c) or FITC-conjugated PPME (d), and fluorescence intensity was analyzed by flow cytometry. Hatched histograms (a-c) represent background fluorescence of cells cultured at 37°C and stained with fluorochrome-labeled isotype-matched control mAb (identical profiles were obtained with 40°C-treated cells). In PPME-binding studies (d), lymphocytes were incubated 30 min at room temperature in PBS alone or with 10 µg/ml of the L-selectin function-inhibiting mAb TQ1. Identical results were obtained over a wide range of serial dilutions of PPME (not shown). Data are representative of four independent experiments.

 
While L-selectin is recognized by mAb reagents regardless of its functional state, the binding capacity of L-selectin for soluble carbohydrate substrates such as PPME can be altered under certain conditions if there is a change in the adhesive potential of this adhesion molecule (24, 25, 30, 35). Therefore, studies were performed to determine the effect of fever-range temperature on PPME binding by lymphocytes. In the study shown in Figure 6God, PBL were incubated for 24 h at 37°C or 40°C, and then L-selectin-mediated adhesion to PPME-FITC was evaluated by flow cytometry in the presence or absence of an L-selectin-blocking mAb, TQ1. Fever-range hyperthermia did not have any detectable effect on L-selectin-specific binding to PPME. Taken together, evidence that fever-range temperatures enhance lymphocyte L-selectin adhesion without affecting overall L-selectin density or recognition of carbohydrate ligands is consistent with the notion that the thermal component of fever enhances L-selectin adhesion and/or avidity at a molecular level.

Effect of hyperthermia on the topographical distribution of L-selectin on PBL

Recent reports have proposed that the preferential localization of L-selectin in focal clusters on surface microvilli in resting human lymphocytes creates concentrated multivalent adhesive domains, thereby increasing the avidity of interactions with low affinity carbohydrate determinants on vascular endothelium (36). Moreover, concentrated presentation of L-selectin on microvillous surface membranes has been proposed to facilitate lymphocyte tethering and rolling on native endothelial ligands under hemodynamic shear forces (1, 2, 36, 37). These observations prompted us to investigate whether the increase in L-selectin adhesion observed in response to fever-range temperature is associated with a change in the topographical distribution of L-selectin on surface membrane domains. To address this question, L-selectin was immunolocalized using DREG-56 mAb and a 20-nm colloidal gold-conjugated secondary Ab, and the surface distribution of gold-labeled L-selectin was examined by TEM. In lymphocytes cultured at 40°C for 24 h, gold particles were predominantly localized on microvillous projections whereas particles were less frequently observed on the planar cell body (Fig. 7GoA). This is the same pattern of L-selectin localization previously described for human PBL and neutrophils (36, 38). Immunogold labeling of L-selectin mAb was not observed above background Ig control levels in PMA-treated lymphocytes that lack surface L-selectin as a result of shedding (1, 2, 25, 28, 39) (data not shown).



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  		FIGURE 7. Hyperthermia increases immunogold labeling of L-selectin on lymphocyte microvilli. A, Human PBL were incubated 24 h at 40°C, and L-selectin was immunolocalized using DREG-56 mAb and a 20-nm colloidal gold-conjugated secondary Ab. The distribution of gold-labeled particles on lymphocyte surface membranes was examined by TEM. Arrows denote gold particles associated with lymphocyte microvilli. B, Quantification of gold particles on distinct plasma membrane regions (i.e, microvilli or cell body) revealed that hyperthermia markedly increased the total number of particles on microvilli as well as the number of particles clustered in groups of five or more. Gold particles were rarely detected on cells that were stained with an isotype-matched negative control mAb.

 
Quantitative analysis of L-selectin topography in PBL cultured either at 37°C or 40°C (Fig. 7GoB) revealed an unexpected finding in light of results indicating that fever-range hyperthermia did not alter the overall L-selectin density on lymphocytes (Fig. 6Goa). In this regard, a marked increase was consistently observed in the total number of gold particles detected on lymphocytes treated at 40°C (Fig. 7GoB). A similar increase in L-selectin labeling of heat-treated cells was detected by flow cytometry using DREG-56 mAb and secondary Ab conjugated to 20-nm fluorescent microspheres (data not shown). The hyperthermia-induced increase in the number of gold particles was restricted to the microvilli and was not observed on the planar cell body. Moreover, fever-range hyperthermia did not cause an obvious change in either the morphology or number of microvilli on lymphocytes or in the level of colloidal gold labeling of other cell surface molecules, i.e., LFA-1 (not shown). Taken together, these data raise the possibility that the increased labeling of L-selectin with colloidal gold-labeled Ab in heat-treated PBL is indicative of a change in the accessibility of this adhesion molecule to gold-labeled Ab, perhaps through alterations in the conformation of L-selectin or proximal molecules in the plasma membrane.

Fever-range hyperthermia stimulates lymphocyte release of soluble factors that enhance L-selectin adhesion

The observed delay of several hours between the initiation of hyperthermia treatment and the increased effect on adhesion suggested that the underlying mechanism involves the synthesis or release of factors that regulate L-selectin adhesion. To determine whether hyperthermia-induced stimulation of adhesion is mediated by soluble factors, lymphocytes were maintained for 24 h at 37°C in the presence of conditioned medium from PBL that had been cultured either at a) 37°C for 24 h or b) 40°C for 12 or 24 h. The results shown in Table IGo demonstrate that nearly equivalent induction of L-selectin-dependent adhesion to HEV occurred when lymphocytes were either cultured continuously at 40°C or maintained at 37°C in the presence of culture supernatants derived from hyperthermia-treated cells. Consistent with the observation that direct exposure to fever-range temperatures did not modulate lymphocyte L-selectin surface density (Fig. 6Goa), culture supernatants from hyperthermia-treated cells similarly had no effect on L-selectin expression (data not shown). These data demonstrate that hyperthermia stimulates lymphocyte release of soluble factors that elicit an increase in L-selectin adhesion to physiologic ligands.


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  		Table I. Fever-range hyperthermia stimulates the release of soluble factors that enhance L-selectin-dependent adhesion of PBL to PLN HEV

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this report, we provide the first evidence that fever-range temperatures act directly at the level of lymphocytes to enhance the function of the L-selectin adhesion molecule, a crucial mediator of lymphocyte emigration into tissues. Specifically, sustained incubation of lymphocytes at 40°C was shown to markedly enhance the ability of human PBL to bind to lymph node HEV in an L-selectin-dependent manner that was calcium dependent and inhibited by mAb specific for L-selectin and PLNAd. Exposure of lymphocytes to fever-range temperatures was further found to increase L-selectin-dependent homing of these cells to PLN, MLN, and PP. In contrast, we were unable to detect a stimulatory effect of 40°C temperature on lymphocyte LFA-1 function, indicating that hyperthermia plays a relatively restricted role in controlling the initial adhesion events required for lymphocyte extravasation. Based on these observations, we propose that fever-range temperatures play an integral role in the generation of an efficient immune response through the amplification of L-selectin-dependent entry of lymphocytes into lymphoid tissues, i.e., lymph node and PP, as well as injured or inflamed tissues.

Several previous studies have implicated the thermal component of fever as a positive activator of lymphocyte immune function. For example, temperatures within the physiologic range of fever have been shown to stimulate lymphocyte proliferative responses to mitogens and allogeneic lymphocytes (6, 10, 11, 12), specific T lymphocyte-mediated cytotoxicity (10, 11, 12), and Ab production (10). The findings of the present study demonstrating that fever-range hyperthermia also enhances lymphocyte L-selectin adhesion provide additional support for the concept that, during infection or injury, the febrile response functions at multiple, cooperative levels to augment host immune defenses. In related studies, we have recently determined that whole body hyperthermia of several hours at fever-level temperatures markedly enhances the ability of lymph node HEV to support L-selectin-mediated lymphocyte adhesion (S. S. Evans, manuscript in preparation). Taken together, evidence that hyperthermia dynamically regulates L-selectin adhesion potential both at the level of lymphocytes and the HEV strongly implicates the thermal component of fever as an important physiologic mechanism to increase lymphocyte recirculation through lymphoid tissues. Moreover, localized temperature elevation within tissues during infection or injury may significantly increase the number of cells that can be recruited specifically to these tissues.

The effect of fever-range temperatures on L-selectin function was found to be highly regulated in a time- and temperature-dependent manner that may reflect important protective mechanisms necessary to maintain immune homeostasis in vivo. The requirement for continuous, extended exposure (i.e, 9 h) of lymphocytes to hyperthermia could prevent fluctuations in adhesion potential that might otherwise occur during lymphocyte recirculation through regions of the body with extreme temperature variations; e.g., core body temperature of humans is ~ 37 to 38°C whereas the skin is <= 32°C (40). Moreover, the finding that removal of the heat stimulus or prolonged (>24 h) exposure to hyperthermia are both associated with a return to normal levels of L-selectin adhesion may reflect an important feedback mechanism to prevent sustained, inappropriate recruitment of leukocytes to tissues. In this regard, during infection or injury, the first 24 h are critical for leukocyte recruitment to tissues whereas, after this period, continued leukocyte extravasation has been associated with severe tissue damage. Thus, normalization of adhesion immediately after recovery from infection or during prolonged febrile responses would appear to have important physiologic significance.

Data presented in this study support the emerging concept that L-selectin adhesion and avidity can be actively regulated at multiple levels. Notably, fever-range hyperthermia was shown to augment L-selectin adhesion without affecting lymphocyte L-selectin surface density or the ability to bind PPME carbohydrate molecules. Although Scatchard analysis of L-selectin binding to PPME carbohydrate substrate was not performed, the failure to detect a change in PPME binding in response to heat by flow cytometric analysis suggests that L-selectin affinity is not influenced by hyperthermia. Thus, elevated temperatures appear to regulate the adhesion potential and/or avidity of pre-existing L-selectin molecules. An unexpected finding was that hyperthermia caused a marked increase in the amount of L-selectin detected on lymphocytes using secondary Ab conjugated to colloidal gold or fluorescent microspheres. One potential explanation for these results is that, in addition to stimulating lymphocyte-HEV adhesion, fever-range temperatures alter the accessibility or conformation of L-selectin, thereby facilitating interactions with large molecules such as Ab conjugated to 20-nm gold particles or microspheres. Although an obvious change in the length or number of microvilli was not observed in hyperthermia-treated cells, these changes have not been formally ruled out in the present study. Thus, the increase in L-selectin labeling by colloidal gold or microsphere particles could be due to changes in microvillous membrane structure, L-selectin protein conformation, or the association of L-selectin with proximal membrane or intracellular proteins.

Several recent studies have similarly noted changes in L-selectin adhesion that are not accompanied by alterations in L-selectin surface levels. Specifically, L-selectin adhesion and/or avidity are reportedly regulated by: a) physical interactions between the L-selectin cytoplasmic domain and the actin-based cytoskeleton (2, 41, 42) (and S. S. Evans, unpublished observations), b) restricted localization of L-selectin on microvillous surface membranes (1, 2, 36, 37, 38, 42), and c) shear forces above a critical threshold that promote L-selectin-dependent rolling interactions of lymphocytes along the luminal surface of vascular endothelium (43). With regard to the latter observation, it is tempting to speculate that stimulation of L-selectin adhesion by hyperthermia represents a compensatory mechanism to offset the suboptimal shear forces that would theoretically occur as a result of fever-induced vasodilation within HEV.

The mechanisms by which hyperthermia regulates L-selectin adhesion and/or avidity remain to be determined. Elevated temperatures reportedly increase plasma membrane fluidity (44), which could influence adhesion molecule clustering and, thereby, avidity for endothelial cell ligands. Moreover, hyperthermia has been shown to alter directly the functional activity of proteins such as IFNs or Ab (6, 10, 13, 14). While these mechanisms may contribute to the regulation of L-selectin function in vivo, several lines of evidence indicate that direct thermal effects on membrane fluidity or L-selectin binding activity are not solely responsible for the increase in L-selectin adhesion observed in vitro. In this regard, since the adhesion assays and immunogold-labeling procedures were performed at 4°C, any direct effects of heat on membrane fluidity or L-selectin activity would likely be negated at this low temperature. In addition, equilibration of lymphocytes at fever-range temperatures for 2 h was not sufficient to maximally stimulate L-selectin-dependent lymphocyte-HEV interactions. Finally, the most compelling evidence was that significant increases in L-selectin-dependent adhesion were observed in lymphocytes that were not exposed to elevated temperatures but were instead cultured with conditioned medium derived from hyperthermia-treated cells.

Studies are in progress to identify the soluble factors that regulate L-selectin adhesion in response to thermal stimuli. Candidate factors include several proinflammatory cytokines such as TNF-{alpha}, IL-1ß, IFN-{gamma}, and IFN-{alpha} that are produced in response to hyperthermia in vitro or in vivo (6, 10, 45, 46, 47, 48). Of particular interest, IFN-{alpha}/ß as well as IFN-inducible proteins have recently been shown to regulate L-selectin synthesis and shedding in human lymphocytes (24, 25), raising the possibility that these cytokines also participate in control of L-selectin avidity. Fever-range temperatures may also stimulate the release of unidentified factors from lymphocyte surface membranes that have adhesion-potentiating activity. In addition, a potential cause-and-effect relationship may exist between the heat-induced enhancement of L-selectin adhesion, described herein, and the previously reported hyperthermia-induced changes in the intracellular localization and expression of functionally important proteins in lymphocytes including heat shock proteins (hsp70), signal transduction proteins such as protein kinase C, and the cytoskeletal protein spectrin (22, 23).

The results of the present study may have important implications in the use of hyperthermia as a treatment modality in cancer patients. Hyperthermia therapy frequently involves short-term (<2h) high temperatures (>=41°C) (48, 49, 50, 51), conditions that would not be expected to augment L-selectin adhesion. However, studies in animal models have suggested that long duration, fever-range whole body hyperthermia (6 h, 40°) has increased efficacy in the treatment of tumors compared with short duration, high temperature therapy (2 h, 41.5°C), particularly in an adjuvant setting in combination with chemotherapy and immunotherapy (52, 53). Recent studies from our group have further shown that fever-range whole body hyperthermia (39.5°C, 9 h) induces an anti-tumor immune response involving, at least in part, NK cells (54). It is noteworthy, in this regard, that fever-range whole body hyperthermia significantly alters several parameters of lymphocyte structure and function that are closely associated with lymphocyte activation (22, 23). Future studies are required to test the hypothesis favored by the present data that therapeutic strategies involving long duration, fever-range temperatures stimulate L-selectin-dependent lymphocyte-endothelial cell interactions, thereby increasing access of immune effector cells to regional lymph nodes and tumor tissues.


   Acknowledgments
 
We thank Drs. Eugene Butcher, Ronald Ward, Lloyd Stoolman, and William Muller for graciously providing the mAb, PPME-FITC reagent, and cell lines used in these studies; Dr. Carleton Stewart of the Flow Cytometry Facility for advice on the flow cytometric analysis; Debbie Ogden, of the Cell Analysis Facility, and Adam Sumlin for technical help; Margaret Frey for assistance in the preparation of the figures; and Dr. Jennifer Black for helpful guidance for TEM and for critical reading of the manuscript.


   Footnotes
 
1 This work was supported in part by National Institutes of Health Grant RR08926 (to S.S.E), Department of Defense Grant 8570607 (to E.A.R.) and Grant GM45994 (to J.R.S.), the Roswell Park Alliance Foundation (S.S.E. and E.A.R.), Cancer Center Support Grant P30 CA16056–21 at Roswell Park Cancer Institute, and the Dr. Louis Sklarow Memorial Fund (S.S.E.). Back

2 Address correspondence and reprint requests to Dr. Sharon S. Evans, Roswell Park Cancer Institute, Buffalo, NY 14263. E-mail address: Back

3 Abbreviations used in this paper: PP, Peyer’s patches; HEV, high endothelial venule; MLN, mesenteric lymph node; MAdCAM-1, mucosal addressin cellular adhesion molecule-1; PLN, peripheral lymph node; PLNAd, PLN addressin; PPME, phosphomonoester core polysaccharide; Ri, ratio of FITC-PKH26-labeled cells before injection; Ro, ratio of FITC-PKH26-labeled cells after injection; TEM, transmission electron microscopy. Back

Received for publication May 29, 1997. Accepted for publication October 1, 1997.


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