Uropod Formation Distribution, Protein Kinase C Activity, And

Effect of Fever-Like Whole-Body Hyperthermia on Lymphocyte Spectrin Distribution, Protein Kinase C Activity, and Uropod Formation This information is current as of September 26, 2021. Xiang-Yang Wang, Julie R. Ostberg and Elizabeth A. Repasky J Immunol 1999; 162:3378-3387; ; http://www.jimmunol.org/content/162/6/3378 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 1999 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Effect of Fever-Like Whole-Body Hyperthermia on Lymphocyte Spectrin Distribution, Protein Kinase C Activity, and Uropod Formation1

Xiang-Yang Wang, Julie R. Ostberg, and Elizabeth A. Repasky2

Regional inﬂammation and systemic fever are hallmarks of host immune responses to pathogenic stimuli. Although the thermal element of fever is thought to enhance the activity of immune effector cells, it is unclear what the precise role of increased body temperatures is on the activation state and effector functions of lymphocytes. We report here that mild, fever-like whole body hyperthermia (WBH) treatment of mice results in a distinct increase in the numbers of tissue lymphocytes with polarized spectrin cytoskeletons and uropods, as visualized in situ. WBH also induces a coincident reorganization of protein kinase C (PKC) isozymes

and increased PKC activity within T cells. These hyperthermia-induced cellular alterations are nearly identical with the previously Downloaded from described effects of Ag- and mitogen-induced activation on lymphocyte spectrin and PKC. Immunoprecipitation studies combined with dual staining and protein overlay assays conﬁrmed the association of PKC␤ and PKC␪ with spectrin following its reorganization. The receptor for activated C kinase-1 was also found to associate with the spectrin-based cytoskeleton. Furthermore, all these molecules (spectrin, PKC␤, PKC␪, and receptor for activated C kinase-1) cotranslocate to the uropod. Enhanced intracellular spectrin phosphorylation upon WBH treatment of lymphocytes was also found and could be blocked by the PKC inhibitor

bisindolylmaleimide I (GF109203X). These data suggest that the thermal element of fever, as mimicked by these studies, can http://www.jimmunol.org/ modulate critical steps in the signal transduction pathways necessary for effective lymphocyte activation and function. Further work is needed to determine the cellular target(s) that transduces the signaling pathway(s) induced by hyperthermia. The Journal of Immunology, 1999, 162: 3378–3387.

pon activation, lymphocytes undergo many morpholog- binding domain (11, 12), an src homology domain 3 (SH3)3 (13), ical and biochemical changes. For example, activated T a pleckstrin homology (PH) domain (14, 15), and a membrane U cells display polarization, with the formation of a well- adhesion domain (16). Because a variety of proteins are known to defined single cytoplasmic projection termed the uropod (1, 2). interact with spectrin, it has been speculated that assembly of the These cells also exhibit a redistribution of adhesion molecules (i.e., spectrin-based skeleton participates within specialized membrane by guest on September 26, 2021 ICAM-1, ICAM-3, and CD44) (3, 4) and cytoskeletal elements, domains and in the construction of cell-cell contacts (17). Thus, including microtubules and ezrin-radixin-moesin family members spectrin is an important molecule to evaluate during lymphocyte (5–7), to the uropod. Our laboratory has previously shown that the activation (18). uropod forms in close association with an aggregate of spectrin We previously characterized a heterogeneous subcellular distri- that develops in response to various activation signals (8, 9). While bution of spectrin in lymphocytes and found that spectrin and other the uropod appears to be important in cell interactions and recruit- cytoskeletal proteins redistribute to distinct polar aggregates and ment of immune effector cells to inflammatory sites, the precise caps upon various methods of lymphocyte activation (9, 18, 19). mechanism and functional role of uropod formation are poorly Spectrin aggregate formation has been shown to coincide with the understood. positioning of the newly formed uropod following lymphocyte ac- The development of cell polarity seems to involve interactions tivation (9).4 Most interestingly, a key signaling molecule, protein between components of the cell membrane and cytoskeleton as kinase C (PKC) ␤II isozyme, also translocates to the spectrin ag- well as reorganization of cytoskeletal elements. As a major com- gregate upon cellular activation (20, 21). This strongly suggests a ponent of the membrane cytoskeleton, spectrin has diverse func- role for the dynamic reorganization of the spectrin-based skeleton tional motifs for protein-protein interactions, including an actin- in PKC-mediated signaling pathways that lead to lymphocyte ac- binding domain (10), a Ca2ϩ-binding domain (11, 12), an ankyrin- tivation and morphological polarization. PKC is known to be involved in the regulation of a diverse range of cell functions, including cell proliferation and differentiation, and maintenance of cell morphology and adhesion (22). At least 12 Department of Immunology, Roswell Park Cancer Institute, Buffalo, NY 14263 subspecies of PKC have been identified in mammalian tissue and Received for publication September 1, 1998. Accepted for publication December categorized into three subclasses based on differences in structure 4, 1998. and cofactor requirements: conventional PKCs (␣, ␤I, ␤II, and ␥), The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported in part by National Institutes of Health Grant CA71599, 3 Abbreviations used in this paper: SH3, src homology 3; PH, pleckstrin homology; Roswell Park Cancer Institute Core Grant CA16056-21 for the Cell Analysis Center, PKC, protein kinase C; RACK, receptor for activated C kinase; WBH, whole-body and a postdoctoral training fellowship from the Cancer Research Institute. hyperthermia; TBS, 10 mM Tris-HCl (pH 7.5) and 150 mM NaCl. 2 Address correspondence and reprint requests to Dr. Elizabeth Repasky, Roswell 4 R. L. Campbell, S. Wright, E. A. Repasky, S. C. Watkins, and M. T. Lotze. Early Park Cancer Institute, Elm and Carlton Streets, Buffalo, NY 14263. E-mail address: spectrin aggregation during naive T cell activation distal to the site of antigen pre- [email protected] senting cell (APC) interaction. Submitted for publication.

novel PKCs (␦, ⑀, ␩, ␪, and ␮) and atypical PKCs (␨, ␫, and ␭) (23). Control mice were kept at room temperature and were subjected to han- Although the importance of PKC activity during lymphocyte ac- dling and temperature measurement manipulations similar to those of tivation has been reported, little is known regarding the localiza- WBH-treated mice. After treatment, the mice were sacrificed, and lym- phoid organs were removed and either imprinted or prepared in single cell tion and role of each PKC isozyme in lymphocytes (24). A group suspensions. For in vitro hyperthermia, culture flasks containing cells were of proteins termed receptors for activated C kinase (RACKs) ap- wrapped tightly with Parafilm (American National Can, Neenah, WI) and pear to be important for increasing PKC phosphorylation of sub- then totally submerged in a 39.5°C water bath (Lab-line Instruments, Mel- strates, presumably by stabilizing the active form of PKC (25). rose Park, IL). Control cells were incubated at 37°C. Furthermore, it was recently suggested that PKC binds to specific Indirect immunofluorescence anchoring proteins located at various subcellular sites, thus providing another mechanism for isozyme-specific regulation (26, For in situ staining, freshly isolated spleen was cut and imprinted on coverslips, fixed in 2% formaldehyde, and permeablized with PBS containing 27). Indeed, a number of signal-transducing proteins have recently 0.2% Triton X-100. When localizing both spectrin and PKCs in the same been found to be associated with the membrane cytoskeleton in- cells, the imprints were first stained with goat anti-chicken ␣ spectrin an- terface (28–32), suggesting an important function of membrane tiserum (diluted 1/400 in PBS) (19, 36), followed by rhodamine-conjugated cytoskeleton organization in the molecular regulation of lympho- donkey anti-goat IgG (diluted 1/200; Miles/ICN, Irvine, CA). The same imprints were then incubated with a 1/50 dilution of either rabbit anti- cyte activation. PKC␤ (Life Technologies, Grand Island, NY) or rabbit anti-PKC␪ (Santa In conjunction with the lymphocyte activation caused by patho- Cruz Biotechnology, Santa Cruz, CA), followed by fluorescein-conjugated logical stimuli or other immunomodulators, another cardinal fea- goat anti-rabbit IgG (diluted 1/200; Miles/ICN). Normal goat and rabbit ture of the host response to infection includes local increases in IgG (Santa Cruz Biotechnology) were used as negative controls. Immuno- temperature at the site of inflammation and systemic fever. The fluorescent results were analyzed with a confocal microscope MRC 600 Downloaded from (Bio-Rad, Hercules, CA). complex immunological, neurological, and biochemical interac- Isolated T lymphocytes were prepared for immunofluorescence staining tions that give rise to fever are becomingly increasingly clear. as previously described (19). Enriched populations of T lymphocytes were However, the function of the resulting increased temperature in obtained by passing lymph node cell suspensions over nylon wool columns immune responses is not understood. This is primarily due to the (37). Ninety percent purification was confirmed using FITC-conjugated anti-Thy1.2 Abs (ICN Biomedicals, Costa Mesa, CA) (19). difficulty in separating the effects that are due to the thermal ele-

ment of fever alone from the myriad other physiological and neu- Subcellular fractionation http://www.jimmunol.org/ rological events that occur during the fever response. While there Ice-cold PBS-washed cells were lysed in 20 mM Tris-HCl (pH 7.5), 150 is no clear proof of a specific cellular effect of this evolutionarily mM NaCl, 0.5 mg/ml digitonin (Sigma, St. Louis, MO), 5 mM MgCl2,2 conserved response to infection (i.e., increased body temperature), mM EDTA, 2 mM EGTA, 250 mM sucrose, 25 ␮g/ml aprotinin, 10 ␮g/ml it is assumed that fever may have the ability to enhance immuno- leupeptin, and 1 mM 4-(2-aminoethyl)benzenesulfonyl fluoride (Alexis, logical functions (33). San Diego, CA). Cytosolic and particulate fractions were separated by sedimentation at 100,000 ϫ g for 50 min at 4°C (TL-100, Beckman, Ful- The use of externally applied heat has previously been used by lerton, CA). The pellets were extracted in 1% Triton X-100 containing lysis our group in attempts to dissect the effects of the thermal element buffer and further spun at 15,600 ϫ g for 20 min, and the supernatants of fever. Application of this fever-like hyperthermia has been (Triton-soluble membrane fraction) were collected. The Triton-insoluble shown to induce multiple changes in lymphocytes that are indic- pellets (cytoskeletal fraction) were resuspended in cytoskeletal buffer by guest on September 26, 2021 (125 mM Tris-HCl (pH 6.8), 1.25% SDS, 2 mM EDTA, 0.25 M sucrose, ative of altered activation levels. These include 1) significant al- 10% glycerol, and 1% 2-ME) and sonicated three times for 5 s each time terations in the organization of the spectrin-based cytoskeleton on ice. Protein content was determined using a BCA assay kit (Pierce, (34), 2) activation and subcellular reorganization of PKC (34), 3) Rockford, IL). induction of heat shock proteins (most notably heat shock proteins Western blot analysis 70 and 110) (34), and 4) increased L-selectin-dependent adhesion of lymphocytes to vascular endothelium (35). Most of these cel- Equivalent protein samples were subjected to 7.5–10% SDS-PAGE (38) lular changes are not seen following the more severe and acute and transferred onto an Immobilon-P membrane (Millipore, Marlborough, MA). Membranes were blocked with 5% nonfat milk in 20 mM Tris-HCl hyperthermia protocols that are usually employed in heat shock (pH 7.4), 137 mM NaCl, and 0.05% Tween-20 for1hatroom temperature protein studies. followed by a 2-h incubation with either of the following primary Abs Here we attempt to characterize further the precise cellular tar- (diluted 1/1000 in 20 mM Tris-HCl (pH 7.4), 137 mM NaCl, and 0.05% gets of fever-like hyperthermia in immune responses and identify Tween-20): rabbit anti-chicken erythrocyte ␣-spectrin serum (19, 36); ␤ mechanisms by which fever may modify lymphocyte function. We mouse monoclonal or rabbit polyclonal anti-PKC (Life Technologies/ BRL); rabbit anti-PKC␣,-␤I, -␤II, -␥,-␦,-⑀,-␪,or-␨ (Santa Cruz Bio- first investigated the pattern of spectrin and PKC isozyme reorga- technology); and mouse anti-RACK1 or mouse anti-PKC␭ (Transduction nization within T lymphocytes in vivo following a fever-like Laboratories, Lexington, KY). After washing, membranes were incubated whole body hyperthermia (WBH) treatment. The potential for in- with horseradish peroxidase-conjugated dog anti-rabbit IgG or dog anti- teractions among spectrin, PKC isozymes, and RACK1 were also mouse IgG (diluted 1/2000; Amersham, Arlington Heights, IL). Immuno- reactivity was detected by the enhanced chemiluminescence detection sys- determined. Lastly, the effects of WBH on PKC activity and spec- tem (Amersham, Arlington Heights, IL). trin phosphorylation were analyzed. Evidence is provided for the involvement of PKC in the dynamic spectrin organization of ac- PKC activity assay tivated lymphocytes. Total and subcellular fractions from T lymphocytes were prepared following WBH treatment and were partially purified with DEAE chromatogra- Materials and Methods phy. PKC activity was measured using the PKC assay system from Life Fever-range hyperthermia treatment Technologies as previously described (39). For assaying PKC activity in spectrin immunoprecipitates, the spectrin immunocomplexes were recov- Eight- to twelve-week-old female BALB/c mice (Springville Laboratories, ered by protein A-agarose beads and washed three times in kinase buffer Springville, NY) were placed in a humidified environmental chamber (20 mM Tris (pH 7.5), 0.5 mM EDTA, 0.5 mM EGTA, 10 ␮g/ml leupeptin, (VWR Scientific, Rochester, NY) that was preheated to 38°C. The incu- and 25 ␮g/ml aprotinin). The kinase assay was performed in a final volume ␮ bator temperature was then increased 1°C every 30 min until mice reached of 50 l of assay solution containing 20 mM Tris (pH 7.5), 20 mM MgCl2, Ϯ ␮ ␮ a core temperature of 39.5 0.3°C. Core temperatures were monitored 1 mM CaCl2,10 M PMA, 0.28 mg/ml phosphatidylserine, 20 M ATP, during WBH using a thermocouple inserted into the rectum and connected and 50 ␮M Ac-myelin basic protein peptide with 10 ␮Ci of [␥-32P]ATP to a digital readout (Sensortek, Clifton, NJ). Mice were maintained at a (ICN Biomedicals)/assay. Incorporation of radioactivity was determined by 39.5°C core temperature in the chamber for various periods up to 6 h. liquid scintillation counting (Packard, Downers Grove, IL). PKC activity 3380 HYPERTHERMIA EFFECTS ON SPECTRIN AND PKC IN LYMPHOCYTES

was expressed as picomoles of phosphate incorporated per minute per 108 cells. Immunoprecipitation Lymphocytes were washed three times with cold PBS and lysed in 10 mM Tris-HCl (pH 7.4), 150 mM NaCl, 2 mM EDTA, 2 mM sodium orthovan- date, 0.5% sodium deoxycholate, 0.1% SDS, 1% Nonidet P-40, 10 ␮g/ml leupeptin, 25 ␮g/ml aprotinin, and 1 mM 4-(2-aminoethyl)benzenesulfonyl fluoride. Cell extracts were preabsorbed with 0.05 vol of preimmune serum together with 30 ␮l of protein A-agarose beads for1hat4°Candcentri- fuged at 1500 rpm for 15 min. The lysate was incubated overnight at 4°C with rabbit anti-chicken spectrin Ab (diluted 1/100). Immune complexes were precipitated with 20 ␮l of protein A-agarose for2hat4°Cand washed twice with 1 ml of wash buffer (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate, and 1% Nonidet P-40), then washed with 50 mM Tris-HCl (pH 6.8) and 0.1% Nonidet P-40 and boiled in 30–40 ␮l of SDS sample buffer before loading on an SDS-PAGE gel. Overlay assay Binding of PKC to spectrin in vitro was analyzed using a modification of a previously described overlay assay (40). Immunoprecipitated spectrin was transferred to a membrane and blocked with 5% milk in TBS (10 mM Downloaded from Tris-HCl (pH 7.5) and 150 mM NaCl). Membranes were incubated with 20 ␮g/ml partially purified PKC fraction in 50 mM Tris-HCl (pH 7.5), 150 ␮ mM NaCl, 10 mg/ml BSA, 50 g/ml phosphatidylserine, 0.1 mM CaCl2, 1 mM calcium, 1 mM EGTA, 10 ␮g/ml leupeptin, 10 ␮g/ml aprotinin, 10 mM ␤-ME, and 10 ␮M PMA (Sigma) for1hatroom temperature. The membrane was washed three times in TBS containing cofactors. Samples

were fixed with 5% formaldehyde in TBS for 20 min at room temperature, http://www.jimmunol.org/ incubated in 2% glycine in TBS for 20 min, washed three times in TBS, and probed with the anti-PKC Abs as described above. FIGURE 1. Dynamic properties of spectrin aggregate formation during In vivo phosphorylation fever-range WBH. Intracellular localization of spectrin in murine spleen tissue by in situ immunofluorescence staining. Compared with the control Isolated T cells were incubated in phosphate-free RPMI medium supple- value (A; bar ϭ 25 ␮m), the percentage of splenocytes with spectrin ag- mented with 5% dialyzed FCS for 30 min and then labeled for 2 h with 400 gregates increased significantly after6hofWBHtreatment (B; bar ϭ 25 ␮ 32 ␮ Ci/ml [ P]orthophosphate. Cultures were treated with 10 M of the PKC ␮m). A high power image was also shown to display the spectrin aggregate inhibitor bisindolylmaleimide I (GF109203X; Alexis Corp.) for 1 h fol- in uropod (C; magnification, ϫ1000; bar ϭ 10 ␮m). lowed by hyperthermia or PMA (100 ng/ml) treatment. Cells were lysed

and precipitated with goat anti-chicken spectrin Abs (diluted 1/100). The by guest on September 26, 2021 resulting immunocomplexes were boiled in SDS sample buffer and elec- trophoresed on 7.5% SDS-PAGE gels. The gels were dried, and radioactive bands were detected by autoradiography using x-ray film (Kodak viously following various activation signals (20), there was an ob- X-OMAT, Eastman Kodak, Rochester, NY) at Ϫ80°C. vious redistribution of spectrin from the Triton X-100-soluble (membrane) fraction to the Triton X-100-insoluble (cytoskeletal) Results fraction. Thus, WBH treatment appears to induce changes in lym- Dynamic spectrin reorganization in WBH-treated T lymphocytes phocyte morphology and spectrin localization that are identical Following a 6-h WBH treatment, we compared the distribution with those seen following Ag- or mitogen-dependent activation or patterns of spectrin in WBH-treated and control spleen tissue im- biochemical stimulation of PKC. prints, where the microenvironmental relationships are preserved compared with those in in vitro cell suspensions. As determined by PKC isozyme distribution and activity in WBH-treated T cells in situ fluorescence staining, most cells exhibited a ring-like, mem- The ability of WBH to induce alterations in the cellular distribu- brane-associated spectrin distribution in untreated spleen tissue tion and solubility of spectrin led us to next explore the intracel- (Fig. 1A). When mice were treated with WBH, the percentages of lular localization patterns of PKC isozymes in T lymphocytes dur- splenocytes with a polar spectrin aggregate and with uropods at the ing WBH. T cells were isolated from the lymph nodes of site of the spectrin aggregate increased significantly (Fig. 1, B and WBH-treated mice at various times. Immunoblot analysis of total C). Furthermore, we observed an increase in the numbers of cell lysates and cell fractions (cytosol, membrane, and cytoskeletal splenocytes where spectrin had formed a distinct cap at one pole of fractions based on detergent solubility) with PKC isozyme-specific the cell. These phenomenon are consistent with the observed pat- Abs revealed the effect of low hyperthermia stress on solubility terns of spectrin rearrangement and uropod formation during lym- changes and subcellular translocation of PKC isozymes (Fig. 3). phocyte activation (8, 9, 19, 20). Fever-range WBH resulted in an Among all PKCs we detected, conventional PKC␣,-␤I, and -␤II; ϳ4-fold increase in the percentage of lymphocytes with uropods novel PKC␪,-⑀, and -␦; and atypical PKC␨ and -␭ were constitu- (Fig. 1C). To directly examine the effects of WBH on the forma- tively expressed in control and WBH-treated murine T lympho- tion of spectrin aggregates in purified T lymphocytes, T cells were cytes. In nonstimulated cells (time zero), these PKCs were present isolated from WBH-treated mice at various time points and stained in the cytosol (␣, ␤I, ␤II, ␪, ⑀, ␦, ␨, and ␭) associated with the with anti-spectrin Ab. After6hofWBHtreatment, the percentage cellular membrane (␣, ␤I, ␤II, ␪, ⑀, ␦, ␨, and ␭) or associated with of T cells with spectrin aggregates or caps had increased from ϳ20 the cytoskeleton (␤I, ␤II, ␦, ␪, and ⑀). Although WBH was not to 55% (Fig. 2A). The effects of WBH on the cellular distribution capable of altering total PKC protein levels, all PKC isozymes of spectrin were also determined by analysis of the solubility prop- displayed an obvious redistribution. PKC␣,-␤II, -␪, and -␭ trans- erties of spectrin (Fig. 2B). No changes in total expression levels located from the cytosol to the membrane fraction, with the highest of spectrin during WBH were observed. However, as shown pre- accumulation at the membrane after 2–4 h of WBH exposure. The Journal of Immunology 3381

The observed selective translocation of PKC isozymes suggests that PKCs are involved in hyperthermia-induced signaling transduction events. Thus, to determine PKC activity in WBH-treated cells, enzymatic assays were performed (Fig. 4). Interestingly, compared with the level in control cells (14.85 Ϯ 1.22 pmol/min/ 108 cells), total PKC activity was found to increase with time of WBH exposure and to reach a maximum level (23.60 Ϯ 1.10 pmol/min/108 cells) after4hofWBHtreatment. Consistent with these results, WBH caused a decrease in PKC activity in the cytosol and simultaneous four- and 3-fold increases in membrane and cytoskeletal fractions, respectively. This increased PKC activity in the cytoskeletal fraction of T cells strongly suggests a role for activated PKC in cytoskeleton function.

Determination of spectrin association with PKC␤ and -␪ Previous ﬁndings of colocalization of spectrin and PKC␤II (20) led us to investigate whether other PKC isozymes could associate ␤ ␤ ␪

with spectrin. It was found that PKC I, - II, and - were present Downloaded from in spectrin immunocomplexes from control, in vitro hyperthermia- treated, and PMA-treated T lymphocytes (Fig. 5), while the other PKC isozymes were not detected (data not shown). Protein overlay assays also revealed the direct association of PKC␤ with immunoprecipitated spectrin from untreated, in vitro hyperthermia- treated, or PMA-treated T lymphocytes in a manner that could be http://www.jimmunol.org/ blocked by the PKC inhibitor GF109203X (data not shown). These data provided evidence that the association between PKC␤ and spectrin is dependent on PKC activation. The direct binding of PKC␪ with spectrin was not detected by this protein overlay method (data not shown). However, this does not eliminate the possibility that PKC␪ might associate with spectrin indirectly through other proteins, such as an unknown anchoring protein. To further explore the localization patterns of spectrin, PKC␤,

and PKC␪ during WBH treatment, we performed in situ double by guest on September 26, 2021 staining of spleen tissue with anti-spectrin and anti-PKC Abs. PKC␤ and spectrin have the same localization patterns in untreated FIGURE 2. Kinetic properties of spectrin aggregate formation in puri- splenocytes (Fig. 6A). When mice were treated with WBH for 6 h, fied T lymphocytes and solubility analysis of spectrin aggregates during fever-range WBH. A, T lymphocytes were isolated from BALB/C mice these two molecules redistributed and colocalized within the ag- ␪ directly after 2, 4, or6hofWBHtreatment (0 h indicates control mice) and gregates (Fig. 6B). Dual staining for PKC and spectrin revealed were stained with anti-spectrin Ab. The percentage of T lymphocytes with the same pattern of redistribution to aggregates (data not shown). spectrin aggregate was quantified by counting a minimum of 200 cells/ Since fever-range WBH treatment appears to induce lymphocyte spectrin aggregation in conjunction with uropod formation, it was ,ء .group. Data are shown as the average values of three experiments Ϯ SE p Ͻ 0.001 compared with the control value (0 h) using unpaired Student’s of interest to determine whether PKC␤ or PKC␪ also colocalized t test. B, Cell fractions were prepared from T lymphocytes isolated directly with spectrin in the uropod of WBH-treated lymphocytes. Double- after 2, 4, or6hofWBHtreatment (0 h indicates control mice), and staining studies showed that both of these PKC isozymes translo- equivalent protein amounts were loaded onto 7.5% SDS-PAGE gel. Spec- cated with spectrin to the uropod following WBH (Fig. 6, C and trin detection was performed with rabbit anti-spectrin serum in both the D), suggesting a role for the PKC/spectrin complex in lymphocyte Triton X-100-soluble (membrane-associated) and Triton X-insoluble (cytoskeleton-associated) fractions with time of WBH. The last lane shows the uropod function. positive control, where spectrin was first immunoprecipitated from control T cells; lower m.w. bands are the rabbit IgG used in the immunoprecipi- Identification of RACK1 in the spectrin-based skeleton tation. The results shown here are representative of three separate Because RACKs appear to be important in PKC localization and experiments. function, we wanted to determine the role of the PKC binding protein RACK1 in the PKC isozyme localization phenomenon described above. Total cell extracts and cellular fractions of T lym- PKC␤I, -⑀,-␦, and -␨ showed no significant reduction in the cy- phocytes from WBH-treated mice were analyzed by immunoblot tosol, indicating that only a small proportion of these PKC using anti-RACK1 Ab (Fig. 7A). Interestingly, RACK1 was seen isozymes translocated to the particulate fraction. Interestingly, the to be constitutively expressed in T lymphocytes and translocated cytoskeletal association of PKC␤I, -␤II, -␪, and -⑀ increased sig- from the cytosol to the membrane and cytoskeleton fractions fol- nificantly during WBH. In addition, double immunoreactive bands lowing WBH. RACK1 was also present in the spectrin immuno- were seen in Western blots for PKC⑀,-␨, and -␭. The upper band precipitates of T cells (Fig. 7B). Compared with control cells, the is believed to be hyperphosphorylated PKC, and the lower band is relative amount of RACK1 protein increased in the spectrin im- believed to be hypophosphorylated PKC (41), indicating possible munoprecipitates when cells were treated with either hyperthermia regulation of PKC activation through autophosphorylation or or PMA in vitro. Furthermore, following WBH treatment, RACK1 phosphorylation by other protein kinases. also localized to the lymphocyte uropod (Fig. 7C). Thus, these data 3382 HYPERTHERMIA EFFECTS ON SPECTRIN AND PKC IN LYMPHOCYTES Downloaded from

FIGURE 4. Assay of PKC activity in T lymphocytes treated by fever- range WBH. Total cell lysates and cytosolic, membrane, and cytoskeletal fractions were prepared directly after 0, 2, 4, and6hofWBHtreatment. PKC was partially puriﬁed from each fraction, and PKC activity was determined as picomoles of phosphate incorporated per minutes per 108 cells. /p Ͻ http://www.jimmunol.org ,ء .Data are shown as the average values of three experiments Ϯ SE 0.05 compared with the control value (0 h) using unpaired Student’s t test.

support a role for RACK1 in the formation and maintenance of cytoskeleton-based morphological changes in stimulated T cells.

Effect of fever-range WBH on intracellular spectrin phosphorylation Previous experiments have shown that pretreatment with PKC in- by guest on September 26, 2021 hibitors reduced the percentage of cells with spectrin aggregates (20). Therefore, we wanted to determine whether spectrin-based skeleton reorganization is affected via phosphorylation by PKC. For this purpose isolated T lymphocytes were labeled with 32P and incubated with or without GF109203X followed by in vitro hyperthermia for6horPMAstimulation for 30 min. Spectrin was immunoprecipitated and analyzed by SDS-PAGE (Fig. 8A). Com- pared with control cells, spectrin from in vitro hyperthermia and PMA-treated cells displayed increased phosphorylation. Pretreat- ment with PKC inhibitor reduced the phosphorylation of spectrin

FIGURE 3. Western blot analysis of PKC isozyme distribution and translocation during fever-range WBH. T lymphocytes were isolated from mice treated with WBH for the indicated times. Cell lysates were separated FIGURE 5. Determination of interaction between spectrin and PKC into cytosolic and particulate fractions, and the particulate fractions were isozymes. A-C, Identification of PKC isozymes in the spectrin immuno- further separated into a membrane fraction (Triton X-100 soluble) and a complex. Spectrin was immunoprecipitated from T lymphocytes before cytoskeletal fraction (Triton X-100 insoluble). The expressions of conven- (lane 1) or after6hofinvitro hyperthermia (lane 2) or 30 min of PMA tional PKCs (A), novel PKCs (B), and atypical PKCs (C) were determined (100 ng/ml) treatment (lane 3). Total cell lysate was used as a positive by immunoblotting using PKC isozyme-specific Abs. Double arrows (e.g., control (lane 4). Immunoblotting was performed using specific Abs for PKC⑀) indicate hyperphosphorylated and hypophosphorylated PKC iso- PKC␤I(A), PKC␤II (B), and PKC␪ (C). The arrow indicates the PKC forms. The results shown here are representative of three separate band; lower m.w. bands are the rabbit IgG used in the immunoprecipita- experiments. tion. These results are representative of three separate experiments. The Journal of Immunology 3383 Downloaded from http://www.jimmunol.org/

FIGURE 6. Colocalization of spectrin with PKC␤ and PKC␪ by double immunoﬂuorescence staining. Spleen imprints were made before (A) and after (B) WBH treatment and incubated with goat anti-spectrin Ab (A and B, left) followed by TRITC-labeled dog anti-goat IgG staining. Coverslips were then incubated with rabbit anti-PKC␤ Abs (A and B, right) followed by FITC-labeled dog anti-rabbit IgG staining (double and single arrowheads indicate spectrin/PKC localizations before and after WBH, respectively; bar ϭ 25 ␮m). T lymphocytes were also isolated from WBH-treated mice and double- stained for spectrin and PKC␤ (C)orPKC␪ (D; the white arrow indicates spectrin/PKC aggregates, and the open arrow indicates localization of spectrin by guest on September 26, 2021 and PKC isozymes at uropod; bar ϭ 10 ␮m).

in response to both in vitro hyperthermia and PMA. To determine during this stage of T cell activation, and that WBH alone can whether the PKC associated with spectrin is an activated form, we enhance these cellular events. Moreover, the presence of spectrin- directly assayed PKC activity in the spectrin immunocomplex associated PKC in the uropod suggests a previously underappre- (Fig. 8B). Compared with the level in control cells (0.54 Ϯ 0.06 ciated role for kinase function in the initiation and/or maintenance pmol/min/108 cells), PKC activity in spectrin immunoprecipitates of this site for cellular interactions. were significantly elevated and reached the highest level after 6 h PKC has been implicated in the activation of the transcription of WBH (2.26 Ϯ 0.22 pmol/min/108 cells). These data are sup- factor activating protein-1 and IL-2 synthesis regulation (24, 47). portive of a hypothesis that PKC is involved in spectrin phosphor- Recently, PKC␪ was found to be translocated to the site of cell ylation and subsequent spectrin reorganization. contact between T cells and APC upon T cell activation (48). In our studies, PKC isozymes (␣, ␤I, ␤II, ␪, ␦, ⑀, ␨, and ␭) in T cells display different distribution patterns, suggesting that an individual Discussion isozyme may mediate distinct signaling regulation. We found that Effective immune responses are heavily dependent on both intra- fever-like WBH resulted in a 2-fold increase in total PKC activity cellular and intercellular events mediated by specialized cell sur- and an increase in membrane- and cytoskeleton-associated PKC face molecules and increased motility and homing. During the mi- activity that is consistent with the translocation pattern of PKC gration of lymphocytes toward an inflammatory site, one of the isozymes (␤I, ␤II, and ␪) during WBH. This may indicate a pos- earliest changes to take place is cellular polarization and uropod sible role for these PKCs in membrane function and cytoskeletal formation (1, 2). While it is not clear whether there is a functional reorganization. It may also be hypothesized that PKC located in role for the uropod that is directly involved in cell motility, uropod the cytosol of resting cells will translocate to the membrane upon formation has been observed during lymphocyte interaction with activation. Other investigators have shown that 42.5°C hyperther- macrophages (42), mitogen or chemokine stimulation of lympho- mia treatment resulted in an elevation of PKC activity (49, 50). In cytes (2, 43), and cell-mediated cytotoxicity (44). Furthermore, the contrast, hyperthermia at 45°C caused a significant decrease in uropod, with its increased concentration of adhesion and effector PKC activity and an increase in activity of phospholipid-indepen- molecules (3, 4, 45, 46), appears to be an important site of cell dent protein kinases (51). However, the observations using hyper- interactions. Our observation of spectrin, PKC␤I, PKC␤II, PKC␪, thermia in this nonphysiological range of 42–45°C, which dramat- and RACK1 redistribution to the uropod suggests that these ele- ically inhibits protein synthesis and causes substantial cell death, ments play an important role in the mechanisms that are involved probably should not be directly compared with our studies using a 3384 HYPERTHERMIA EFFECTS ON SPECTRIN AND PKC IN LYMPHOCYTES

FIGURE 7. Subcellular localization of RACK1 in T lymphocytes during fever-range hyperthermia. A, Total cell lysates and cell fractions were prepared Downloaded from from T lymphocytes isolated directly after 0, 2, 4, and 6 h of WBH treatment. Equivalent protein amounts were loaded onto a 12% SDS-PAGE gel and detected with a 1/5000 dilution of anti-RACK1 mAb. B, Spec- trin was immunoprecipitated from control cells (lane 2), WBH-treated (lane 1), or PMA-treated (lane 3)T

cells. Total cell lysate was used as a positive control http://www.jimmunol.org/ (lane 4). RACK1 was detected in all lanes with RACK1 Ab. C, Isolated T lymphocytes from WBH- treated mice were stained with RACK1 Ab (the arrow shows the localization of RACK1 to the uropod; bar ϭ 10 ␮m). The results in A, B, and C are each representative of three separate experiments. by guest on September 26, 2021

much milder fever-like hyperthermia treatment, where average some other linker protein. The positioning of PKCs at specific temperatures are Ͻ40°C for a significantly longer duration. intracellular locations is probably central to its ability to respond Consistent with our previous work (20), immunoprecipitation efficiently to second messengers and to have ready access to sub- studies showed that PKC␤II as well as PKC␤I and PKC␪ are de- strates (27). We speculate that PKC␤I, -␤II, and -␪ associate with tected in the spectrin immunocomplex, implying that these three spectrin and mediate either the reorganization of the spectrin cy- PKC isozymes (␤I, ␤II, and ␪) may play an important role in toskeleton or the phosphorylation of their specific nearby targets spectrin reorganization. Furthermore, protein overlay assays that are involved in T lymphocyte activation. showed the direct association between spectrin and PKC␤, sug- Although PKC inhibitors blocked the spectrin phosphorylation gesting a possible PKC binding site in spectrin. Application of mediated by PKC, we cannot rule out the possibility that other GF109203X in overlay assays abolished this binding process com- protein kinases might also phosphorylate spectrin in response to pletely. GF109203X has been shown to inhibit PKC activity ex- hyperthermia. Based on the observation that the PKC inhibitor clusively via the ATP binding site (52), indicating a requirement calphostin C blocks formation of the spectrin aggregate in re- for PKC activation in this association. Overlay assays did not show sponse to PMA treatment (20) and WBH (data not shown), it the direct binding between spectrin and PKC␪. However, dual seems likely that spectrin participates in membrane cytoskeleton staining showed that PKC␪ colocalized with spectrin in T lym- organization following phosphorylation. We speculate that spec- phocytes, suggesting that PKC␪ might bind to spectrin through trin aggregate formation involves PKC activation and is regulated The Journal of Immunology 3385

Another group of anchoring proteins, RACKs, has been pro- posed to be critically required for the translocation and subsequent function of PKC (59). Our ﬁndings indicate that RACK1 is present at high levels in T lymphocytes and exhibits a similar translocation from cytosol to membrane and cytoskeleton during WBH as some PKC isozymes. RACK1 is also a component of the spectrin immunoprecipitates, and the amount of RACK1 in the spectrin complex increases following WBH and PMA treatment. Furthermore, RACK1 localizes at the uropod induced by WBH. All these data are consistent with the hypothesis that following WBH, RACK1 binds PKC␤ and PKC␪, then associates with spectrin and assem- bles into an aggregate. Because the PH domain can bind to WD40 repeats within the ␤-subunit of G proteins (60, 61), it is of interest to note that RACK1 is a homologue of the G protein ␤-subunit, containing seven WD40 repeat elements (25). Thus, we speculate that spectrin might interact either directly with PKC or with RACK via its PH domain. However, the detailed molecular interaction of PKCs and their receptors with spectrin awaits further investigation. The data presented here bring to light a hitherto largely ignored Downloaded from potential regulator of lymphocyte activation, i.e., core body temperature. We have shown here that external application of fever- like WBH alone, without any other exogenous immunological stimulant, results in PKC activation and reorganization of the spectrin-based cytoskeleton and its associated molecules. These data

imply the existence of an as yet undefined cellular target(s) that is http://www.jimmunol.org/ sensitive to minor changes in body temperature and is capable of interacting with signal transduction pathways involving PKC and the cytoskeleton. Spectrin-associated PKC isozymes (␤I, ␤II, and ␪) are likely to be involved in this spectrin reorganization through a phosphorylation event. The discovery of RACK1 in the spectrin immunocomplex provides a possible mechanism for PKC translocation and association with spectrin. Thus, it is suggested that PKC/RACK1 interact and assemble into a signaling complex, with the cytoskeleton providing a scaffold on which signal proteins and by guest on September 26, 2021 FIGURE 8. Effects of fever-range hyperthermia on PKC-mediated adhesion molecules function. Further investigations are necessary spectrin phosphorylation. A, Isolated T lymphocytes were labeled with to gain an overall understanding of the functional significance of [32P]orthophosphate for 2 h, then cultures were treated with6hofhyper- thermia (lanes 2 and 3) or 30 min of PMA (lanes 4 and 5) in the presence spectrin/PKC/RACK redistribution during fever-range WBH. (lanes 2 and 4) or the absence (lanes 3 and 5) of the PKC inhibitor However, the morphological and structural changes that are in- GF109203X. Lane 1, Untreated purified T cells as a positive control. Spec- duced in lymphocytes by WBH are indicative of increased polar- trin was immunoprecipitated from cell lysates and loaded onto a 7.5% ity, activation, and motility. We speculate that this increased level SDS-PAGE gel. The arrow shows the intracellular protein with apparent of lymphocyte activation would, in turn, increase the efficiency of increased phosphorylation. The results shown here are representative of Ag-dependent interactions, resulting in enhancement of immune two separate experiments. B, T cells were isolated from mice directly after responses. 0,2,4,and6hofWBHtreatment. Spectrin was immunoprecipitated, and Increased body temperature, which occurs naturally during an PKC activity in the immunocomplex was measured using the PKC assay infection, has been previously suggested to enhance the immune system from Life Technologies. Values are shown as the mean Ϯ SE of response in a way that could benefit clinical practice (33). Even in ,ء .triplicate samples and are representative of three separate experiments p Ͻ 0.01 compared with the control value (0 h) using unpaired Student’s cold-blooded animals (i.e., fish and lizards) a behaviorally induced t test. increase in body temperature has been shown to be completely correlated to survival following various infections (62–64). This strongly supports the hypothesis that there is a fundamental im- by PKC-mediated phosphorylation. Spectrin-based skeleton reor- munological effect of increased body temperature. In fact, the use ganization would then provide a framework for efficient PKC of externally applied hyperthermia in cancer therapies has been phosphorylation of its specific substrates shown to potentiate the immune response to various cytokines Others have shown that PKC isozymes bind to specific cytoskel- (65–67), increasing ICAM-1 expression and adhesion of lympho- etal elements. For example, both PKC␤II and PKC⑀ bind to actin cytes to endothelial cells (35, 68). Furthermore, the combination of (53, 54), and PKC⑀ also binds 14-3-3 proteins (55). PKC␣ colo- IL-2 and local hyperthermia abrogated the growth of tumors in calizes with vinculin and talin (56), while PKC␦ associates with mice better than either modality alone (69) and increased traffick- vimentin filaments upon cellular activation (57). PKC␣ and -␤II ing of activated lymphocytes to the tumor area (70). In contrast to have been shown to bind a multienzyme scaffold protein, AKAP conventional high temperature, short duration WBH protocols, 79, which also binds protein kinase A and the Ca2ϩ/calmodulin- where core body temperatures are raised to ϳ41.8–42°C for any- dependent phosphatase (58). These findings have lead to the sug- where from 30 min to 2 h, our laboratory has used a lower tem- gestion that the specific localization of each isozyme by interaction perature, long duration WBH protocol that does not elicit the same with its binding proteins may be a critical regulatory step in the cytotoxicity of normal tissue that can reduce therapeutic gain. In- selection of the substrate to be phosphorylated. deed, the fever-range WBH was found to induce little damage to 3386 HYPERTHERMIA EFFECTS ON SPECTRIN AND PKC IN LYMPHOCYTES living cells while maintaining the ability to cause significant tumor 26. Newton, A. C. 1996. Protein kinase C: ports of anchor in the cell. Curr. Biol. growth delay (71). These data combined with the findings reported 6:806. 27. Newton, A. C. 1997. Regulation of protein kinase C. Curr. Opin. Cell Biol. here on the effects of fever-like WBH on the dynamic properties of 9:161. spectrin and PKC isozymes in T cells have important implications 28. Burridge, K., and M. Chrzanowska-Wodnicka. 1996. Focal adhesions, contrac- tility, and signaling. Annu. Rev. Cell Dev. Biol. 12:463. for the use of WBH in a clinical setting, particularly one involving 29. Tsukita, S., and S. Yonemura. 1997. ERM (ezrin/radixin/moesin) family: from immunotherapy. cytoskeleton to signal transduction. Curr. Opin. Cell Biol. 9:70. 30. Anderson, J. M. 1996. Cell signalling: MAGUK magic. Curr. Biol. 6:382. 31. Sohn, R. H., and P. J. Goldschmidt-Clermont. 1994. Profilin: at the crossroads of Acknowledgments signal transduction and the actin cytoskeleton. BioEssays 16:465. 32. Carraway, K. L., and C. A. 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