Non-redundant requirement for CXCR3 signalling during tumoricidal T-cell trafficking across tumour vascular checkpoints.

T-cell trafficking at vascular sites has emerged as a key step in antitumour immunity. Chemokines are credited with guiding the multistep recruitment of CD8(+) T cells across tumour vessels. However, the multiplicity of chemokines within tumours has obscured the contributions of individual chemokine receptor/chemokine pairs to this process. Moreover, recent studies have challenged whether T cells require chemokine receptor signalling at effector sites. Here we investigate the hierarchy of chemokine receptor requirements during T-cell trafficking to murine and human melanoma. These studies reveal a non-redundant role for Gαi-coupled CXCR3 in stabilizing intravascular adhesion and extravasation of adoptively transferred CD8(+) effectors that is indispensable for therapeutic efficacy. In contrast, functional CCR2 and CCR5 on CD8(+) effectors fail to support trafficking despite the presence of intratumoral cognate chemokines. Taken together, these studies identify CXCR3-mediated trafficking at the tumour vascular interface as a critical checkpoint to effective T-cell-based cancer immunotherapy.

Ex vivo analysis of topotecan: advancing the application of laboratory-based clinical therapeutics.

Topotecan is currently approved for relapsed small-cell lung cancer and ovarian cancer. Topotecan's efficacy in the second-line setting and novel mechanism of action suggest broad antitumour activity. We utilised a clinically validated, cell-death, ex vivo assay in human tumour explants to examine the activity profile of topotecan alone and in combination with other antitumour agents. Serial dilutions of topotecan alone and in combination with other cytotoxic agents were applied to biopsy specimens of non-small-cell lung cancer (NSCLC) and breast, colon, and prostate cancers. Dose-response curves were interpolated to provide 50% lethal concentrations (LC(50)). The degree of synergy (by median effect) and normalised Z-scores (raw scores converted to relative activity distributed around the mean) were then computed. Single-agent activity was observed for topotecan in all four tumour types. In 57 chemotherapy-naive specimens, NSCLC revealed the highest activity, demonstrated by the lowest LC(50) value (0.26+/-0.06 microg ml(-1); P=0.002). Overall, previously treated and chemotherapy-naive specimens revealed no significant differences in mean LC(50)'s. Synergy was observed for several combinations, including topotecan plus cisplatin in prostate and for topotecan plus 5-fluorouracil in breast cancers. The Z-score analyses conducted suggest activity for previously unexplored drug regimens, including topotecan plus 5-fluorouracil, vinorelbine, and mitomycin-C in NSCLC and breast cancer. Phase II studies are underway to determine the degree to which these ex vivo findings will translate into improved clinical results.

Role of cytokines in photodynamic therapy-induced local and systemic inflammation.

Photodynamic therapy (PDT) of tumour results in the rapid induction of an inflammatory response that is considered important for the activation of antitumour immunity, but may be detrimental if excessive. The response is characterised by the infiltration of leucocytes, predominantly neutrophils, into the treated tumour. Several preclinical studies have suggested that suppression of long-term tumour growth following PDT using Photofrin((R)) is dependent upon the presence of neutrophils. The inflammatory pathways leading to the PDT-induced neutrophil migration into the treated tumour are unknown. In the following study, we examined, in mice, the ability of PDT using the second-generation photosensitiser 2-[1-hexyloxyethyl]-2-devinyl pyropheophorbide-a (HPPH) to induce proinflammatory cytokines and chemokines, as well as adhesion molecules, known to be involved in neutrophil migration. We also examined the role that these mediators play in PDT-induced neutrophil migration. Our studies show that HPPH-PDT induced neutrophil migration into the treated tumour, which was associated with a transient, local increase in the expression of the chemokines macrophage inflammatory protein (MIP)-2 and KC. A similar increase was detected in functional expression of adhesion molecules, that is, E-selectin and intracellular adhesion molecule (ICAM)-1, and both local and systemic expression of interleukin (IL)-6 was detected. The kinetics of neutrophil immigration mirrored those observed for the enhanced production of chemokines, IL-6 and adhesion molecules. Subsequent studies showed that PDT-induced neutrophil recruitment is dependent upon the presence of MIP-2 and E-selectin, but not on IL-6 or KC. These results demonstrate a PDT-induced inflammatory response similar to, but less severe than obtained with Photofrin((R)) PDT. They also lay the mechanistic groundwork for further ongoing studies that attempt to optimise PDT through the modulation of the critical inflammatory mediators.

A phase I study of fever-range whole body hyperthermia (FR-WBH) in patients with advanced solid tumours: correlation with mouse models.

Various studies in animal tumour models have revealed the potential of fever-range whole body hyperthermia (FR-WBH) to be used in cancer therapy. To determine the safety of FR-WBH treatment in the clinic, patients with advanced solid tumours were heated in the outpatient setting to 39-39.5 degrees C for 3 or 6h, or 39.5-40 degrees C for 6h using the Heckel-HT 2000 apparatus. These WBH treatments were well tolerated, with no significant adverse events related to cardiac, hepatic, renal or pulmonary systems. In the majority of patients, flow cytometric analysis of peripheral blood leukocyte populations indicated that there were transient decreases in the number of circulating T lymphocytes and a concomitant decrease in the number of L-selectin positive lymphocytes in the peripheral blood. These findings closely mimic the affects seen previously in pre-clinical murine studies in which this same fever-like treatment was shown to inhibit tumour growth. These studies have established the safety of this treatment and will allow for future clinical trials where application of FR-WBH treatment can be combined with other anti-cancer therapies, including immunotherapy and chemotherapy.

Cytokine and adhesion molecule expression in primary human endothelial cells stimulated with fever-range hyperthermia.

Migration of blood-borne lymphocytes into lymphoid tissues and sites of inflammation is initiated by vascular adhesion molecules and proinflammatory cytokines. Previous in vivo studies have shown that febrile temperatures dynamically stimulate adhesion in differentiated high endothelial venules (HEV), which are portals for lymphocyte extravasation. This report examines the direct effect of fever-range hyperthermia on the expression of adhesion molecules and cytokines by primary cultured endothelial cells. In both macrovascular (HUVEC) and microvascular (HMVEC) endothelial cells, fever-range hyperthermia (40 degrees C for 6-12 h) did not affect expression of adhesion molecules (ICAM-1, E-selectin, VCAM-1, P-selectin, PECAM-1, PNAd, MAdCAM-1), cytokine release (IL-1beta, TNF-alpha, IFN-gamma, IL-6, IL-11, IL-12, IL-13), or chemokine secretion (IL-8, RANTES, MCP-1, MIP-1beta, MIG). This is in contrast to the stimulatory effects of TNF-alpha or 43 degrees C heat shock. However, a novel role for fever-range hyperthermia was identified in augmenting actin polymerization in cultured endothelial cells and enhancing the ability of endothelial-derived factors to transactivate the alpha4beta7 integrin lymphocyte homing receptor. These findings provide insight into the tightly regulated effects of fever-range hyperthermia that exclude induction of adhesion in non-activated endothelium of normal blood vessels. Through these mechanisms, it is proposed that febrile temperatures associated with infection or clinical hyperthermia avoid the unproductive exodus of lymphocytes to non-involved extralymphoid tissues while simultaneously promoting lymphocyte delivery to sites of immune activation.

Fever-range hyperthermia dynamically regulates lymphocyte delivery to high endothelial venules.

Fever is associated with increased survival during acute infection, although its mechanism of action is largely unknown. This study found evidence of an unexpectedly integrated mechanism by which fever-range temperatures stimulate lymphocyte homing to secondary lymphoid tissues by increasing L-selectin and alpha4beta7 integrin-dependent adhesive interactions between circulating lymphocytes and specialized high endothelial venules (HEV). Exposure of splenic lymphocytes in vivo to fever-like whole-body hyperthermia (WBH; 39.8 +/- 0.2 degrees C for 6 hours) stimulated both L-selectin and alpha4beta7 integrin-dependent adhesion of lymphocytes to HEV under shear conditions in lymph nodes and Peyer patches. The adhesiveness of HEV ligands for L-selectin and alpha4beta7 integrin (ie, peripheral lymph node addressin and mucosal addressin cell adhesion molecule-1) also increased during WBH or febrile responses associated with lipopolysaccharide-induced or turpentine-induced inflammation. Similar increases in HEV adhesion occurred during hyperthermia treatment of lymph node and Peyer patch organ cultures in vitro, indicating that the local lymphoid tissue microenvironment is sufficient for the hyperthermia response. In contrast, WBH did not augment adhesion in squamous endothelium of nonlymphoid tissues. Analysis of homing of alpha4beta7(hi) L-selectin(lo) murine TK1 cells and L-selectin(hi) alpha4beta7 integrin-negative 300.19/L-selectin transfectant cells showed that fever-range temperatures caused a 3- to 4-fold increase in L-selectin and alpha4beta7 integrin-dependent trafficking to secondary lymphoid tissues. Thus, enhanced lymphocyte delivery to HEV by febrile temperatures through bimodal regulation of lymphocyte and endothelial adhesion provides a novel mechanism to promote immune surveillance.

Gemcitabine plus cisplatin repeating doublet therapy in previously treated, relapsed breast cancer patients.

PURPOSE: To determine the safety and efficacy of gemcitabine plus cisplatin for patients with relapsed adenocarcinoma of the breast. PATIENTS AND METHODS: Previously treated patients with adenocarcinoma of the breast received cisplatin (30 mg/m(2)) plus gemcitabine (1,000 mg/m(2)) on days 1, 8, and 15 of each 28-day cycle, which was changed after patient no. 12 to cisplatin (30 mg/m(2)) plus gemcitabine (750 mg/m(2)) days 1 and 8 of each 21-day cycle. RESULTS: Of 30 patients, three (10%) had complete and 12 (40%) had partial responses, for an overall response rate of 50%. Two objective responses were observed among the four patients accrued after relapse that followed high-dose/stem-cell therapies. The median time to progression was 14 weeks. The median time to progression for objective responders was 23.5 weeks, with a range of 8 to 68 weeks. Toxicities included grades III and IV neutropenia in 13%, anemia in 6%, thrombocytopenia in 31%, grade III nausea in 4%, and grade II peripheral neuropathy in 2% of 151 treatment cycles. Moderate alopecia occurred in four patients. There were no treatment-related deaths. CONCLUSION: Cisplatin plus gemcitabine is active and tolerable for patients with relapsed breast cancer. Responses observed in previously treated patients, including high-dose/stem-cell failures, indicate activity in otherwise drug-refractory patients.

Fever-range hyperthermia stimulates alpha4beta7 integrin-dependent lymphocyte-endothelial adhesion.

Migration of blood-borne lymphocytes into lymphoid tissues is initiated by the L-selectin and alpha4beta7 integrin adhesion molecules. Previous studies have shown that L-selectin adhesion is dynamically regulated by febrile temperatures. It is now reported that fever-range hyperthermia also acts directly on lymphocytes to enhance selected adhesive functions of alpha4beta7 integrin. Fever-range hyperthermia treatment in vitro (40 degrees C, 12 h) of murine TK1 lymphoma cells and human peripheral blood lymphocytes (PBL) stimulates alpha4beta7 integrin-dependent adhesion to high endothelial venules (HEV) in Peyer's patch and mesenteric lymph node frozen sections. TK1 cells are alpha4beta7hi L-selectin(lo), allowing for the analysis of alpha4beta7 integrin without contributions from L-selectin. Adhesion was further shown to involve alpha4beta7 integrin and its endothelial counter-receptor, mucosal addressin cell adhesion molecule-1 (MAdCAM-1) using function-blocking antibodies (i.e. DATK32, HP2/1, MECA-367). Fever-range hyperthermia also promotes alpha4beta7 integrin-mediated aggregation of TK1 cells. In sharp contrast, hyperthermia fails to increase alpha4beta7 integrin adhesion to fibronectin by TK1 cells. Expression of the alpha4beta7 heterodimer on TK1 cells or human PBL is not altered by hyperthermia, suggesting that hyperthermia stimulates adhesion by enhancing alpha4beta7 integrin avidity rather than its cell surface density. These results provide a mechanism whereby febrile temperatures during infection or clinical hyperthermia potentially amplify the immune response by stimulating L-selectin and alpha4beta7 integrin-dependent homing of immune effector cells to lymphoid tissues.

Dynamic association of L-selectin with the lymphocyte cytoskeletal matrix.

L-selectin mediates lymphocyte extravasation into lymphoid tissues through binding to sialomucin-like receptors on the surface of high endothelial venules (HEV). This study examines the biochemical basis and regulation of interactions between L-selectin, an integral transmembrane protein, and the lymphocyte cytoskeleton. Using a detergent-based extraction procedure, constitutive associations between L-selectin and the insoluble cytoskeletal matrix could not be detected. However, engagement of the L-selectin lectin domain by Abs or by glycosylation-dependent cell adhesion molecule-1, an HEV-derived ligand for L-selectin, rapidly triggered redistribution of L-selectin to the detergent-insoluble cytoskeleton. L-selectin attachment to the cytoskeleton was not prevented by inhibitors of actin/microtubule polymerization (cytochalasin B, colchicine, or nocodozole) or serine/threonine and tyrosine kinase activity (staurosporine, calphostin C, or genistein), although L-selectin-mediated adhesion of human PBL was markedly suppressed by these agents. Exposure of human PBL or murine pre-B transfectants expressing full-length human L-selectin to fever-range hyperthermia also markedly increased L-selectin association with the cytoskeleton, directly correlating with enhanced L-selectin-mediated adhesion. In contrast, a deletion mutant of L-selectin lacking the COOH-terminal 11 amino acids failed to associate with the cytoskeletal matrix in response to Ab cross-linking or hyperthermia stimulation and did not support adhesion to HEV. These studies, when taken together with the previously demonstrated interaction between the L-selectin cytoplasmic domain and the cytoskeletal linker protein alpha-actinin, strongly implicate the actin-based cytoskeleton in dynamically controlling L-selectin adhesion.

Differential expression and function of L-selectin on CD56bright and CD56dim natural killer cell subsets.

NK cells are the first line of defense against foreign cells, virally infected cells, and tumors. The mechanisms whereby NK cells accumulate in extralymphoid sites in response to pathogenic stimuli are not well understood. The L-selectin adhesion molecule (CD62L) plays a primary role in mediating the initial interaction of leukocytes with vascular endothelium, a crucial step in the extravasation of immune effector cells into tissues. In this report, we show L-selectin to be uniquely expressed on a subset of resting human NK cells (CD56bright). Notably, CD56bright NK cells expressed L-selectin at a higher density than all other peripheral blood leukocytes. NK activation by PMA, IL-2, IL-15, or TGF-beta down-regulated L-selectin on the CD56bright subset, while increased L-selectin levels were observed in both the CD56bright and CD56dim NK subsets in response to IL-12, IL-10, or IFN-alpha. Moreover, CD56bright NK cells bound with high efficiency to physiologic L-selectin ligands on peripheral lymph node high endothelial venules (HEV). In sharp contrast, CD56dim NK cells adhered poorly to HEV and were predominantly L-selectin- or expressed L-selectin only at low density. In CD56bright cells and a subpopulation of CD56dim cells, L-selectin ligation by mAb cross-linking activated lymphocyte function-associated Ag 1 (LFA-1), a second adhesion molecule required for leukocyte extravasation. LFA-1 was expressed on both NK subsets, although its density was constitutively higher on CD56dim cells. Taken together, evidence of differential expression of L-selectin and LFA-1 on CD56bright and CD56dim NK subsets strongly suggests unique migratory properties and functions of these cells during the early immune response to foreign pathogens.

Fever-range hyperthermia enhances L-selectin-dependent adhesion of lymphocytes to vascular endothelium.

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 degrees 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.

Tyrosine kinase-dependent regulation of L-selectin expression through the Leu-13 signal transduction molecule: evidence for a protein kinase C-independent mechanism of L-selectin shedding.

The L-selectin adhesion molecule mediates lymphocyte extravasation in peripheral lymph nodes, and has also been implicated in directing leukocyte recruitment to inflammatory tissues and metastasis of lymphoid malignancies. In this study, we demonstrate a novel level of regulation of L-selectin expression that involves the 16-kDa Leu-13 signal transduction molecule. Leu-13 is a member of a multimeric cell surface complex in lymphocytes that includes TAPA-1 (target of antiproliferative Ab-1, CD81) as well as lineage-specific proteins. In the present study, mAb-induced ligation of Leu-13 was shown to rapidly down-regulate L-selectin surface density on normal and malignant human lymphocytes, and to markedly inhibit L-selectin-mediated adhesion of lymphocytes to soluble carbohydrate ligands (i.e., PPME, phosphomonoester core polysaccharide) and to lymph node high endothelial venules. Through the use of genistein and staurosporine, potent inhibitors of tyrosine kinases (TK) and protein kinase C (PKC), respectively, Leu-13-induced L-selectin down-modulation was demonstrated to involve a TK-dependent, PKC-independent pathway, and was attributed to increased L-selectin shedding from surface membranes. Notably, direct L-selectin ligation, modeling cross-linking interactions with endothelial cell ligands, similarly down-regulates L-selectin surface expression through a TK-dependent, PKC-independent mechanism. In sharp contrast, PMA and anti-CD3 mAb down-regulate L-selectin via a staurosporine-sensitive, genistein-resistant pathway that is closely linked to lymphocyte proliferation. Taken together, these results demonstrate a novel role for Leu-13- and L-selectin-induced TK activity in control of L-selectin expression, thus providing insight into the complex molecular mechanisms that potentially regulate L-selectin-dependent lymphocyte homing in vivo.

Protein kinase C isozyme-mediated cell cycle arrest involves induction of p21(waf1/cip1) and p27(kip1) and hypophosphorylation of the retinoblastoma protein in intestinal epithelial cells.

The molecular mechanisms underlying protein kinase C (PKC) isozyme-mediated control of cell growth and cell cycle progression are poorly understood. Our previous analysis of PKC isozyme regulation in the intestinal epithelium in situ revealed that multiple members of the PKC family undergo changes in expression and subcellular distribution precisely as the cells cease proliferating in the mid-crypt region, suggesting that activation of one or more of these molecules is involved in negative regulation of cell growth in this system (Saxon, M. L., Zhao, X., and Black, J. D. (1994) J. Cell Biol. 126, 747-763). In the present study, the role of PKC isozyme(s) in control of intestinal epithelial cell growth and cell cycle progression was examined directly using the IEC-18 immature crypt cell line as a model system. Treatment of IEC-18 cells with PKC agonists resulted in translocation of PKC alpha, delta, and epsilon from the soluble to the particulate subcellular fraction, cell cycle arrest in G1 phase, and delayed transit through S and/or G2/M phases. PKC-mediated cell cycle arrest in G1 was accompanied by accumulation of the hypophosphorylated, growth-suppressive form of the retinoblastoma protein and induction of the cyclin-dependent kinase inhibitors p21(waf1/cip1) and p27(kip1). Reversal of these cell cycle regulatory effects was coincident with activator-induced down-regulation of PKC alpha, delta, and epsilon. Differential down-regulation of individual PKC isozymes revealed that PKC alpha in particular is sufficient to mediate cell cycle arrest by PKC agonists in this system. Taken together, the data implicate PKC alpha in negative regulation of intestinal epithelial cell growth both in vitro and in situ via pathways which involve modulation of Cip/Kip family cyclin-dependent kinase inhibitors and the retinoblastoma growth suppressor protein.

Modulation of retinal endothelial barrier in an in vitro model of the retinal microvasculature.

The prediabetic/diabetic condition functionally alters the microvascular bed of the eye and the breakdown in the transvascular barrier may be produced by changes in the retinal endothelial barrier. To better understand how retinal microvessel barrier is maintained and is altered in vivo this study applies and extends our previously described in vitro permeability technique to study retinal endothelial monolayers. The model of the retinal microvasculature consists of retinal capillary endothelial cells cultured on porous microcarrier beads and perfused in chromatographic 'cell-columns'. This model design relies on indicator-dilution techniques to measure the permeability of the retinal endothelial monolayer and detects small changes in retinal endothelial permeability produced by treatments. Bovine retinal capillary endothelial cells (RCE) were obtained using an endothelial selective media. RCE were seeded at 3 x 10(4) cells cm-2 of fibronectin-coated gelatin microcarriers. After 7 days of microcarrier culture, microcarriers were poured to form columns 0.66 cm in diameter and 1.6 cm in length. The cell-column elution patterns of coinjected optically absorbing tracers (blue dextran 2 x 10(6) Da; cyanocobalamin 1355 Da; sodium fluorescein 376 Da) were analysed to estimate the permeability of the RCE monolayers covering the microcarriers. Scanning electron microscopic examination showed complete monolayer formation on the surface of the microcarriers. We found that baseline monolayer permeability averaged 7.57 +/- 0.57 x 10(-5) cm sec-1 for cyanocobalamin and 9.29 +/- 0.78 x 10(-5) cm sec-1 for sodium fluorescein (mean +/- S.E.M., n = 39). Permeability did not increase over 2 hr of cell-column perfusion. Permeability was decreased by 1 micron isoproterenol (n = 3) and increased by 1 microgram ml-1 cytochalasin D (n = 5). This is one of the first reports of in vitro permeability values for the transport barrier formed by retinal microvascular endothelial cells. Furthermore, the endothelial component of the retinal barrier is dynamic, and is enhanced by isoproterenol and diminished by cytochalasin D.

Expression cloning of an interferon-inducible 17-kDa membrane protein implicated in the control of cell growth.

Interferon-inducible membrane proteins of approximately 17 kDa have been suggested to play a role in the antiproliferative activity of interferons based on (1) their pattern of induction in interferon-sensitive and -resistant cell lines and (2) the ability of a membrane fraction enriched in 17-kDa proteins to inhibit cell growth. To gain insight into the nature of the proteins that mediate the antiproliferative activity of interferons, a monoclonal antibody, 13A5, was generated that reacted specifically with a 17-kDa interferon-inducible cell surface protein. The expression pattern of this 17-kDa protein by human cell lines correlated with sensitivity to the antiproliferative activity of interferons. To obtain information regarding the structure of this protein, the 13A5 antibody was used to screen COS cells transfected with a human cDNA expression library. Sequence analysis of a full-length cDNA clone revealed identity with the 9-27 cDNA, previously isolated on the basis of its interferon inducibility by differential screening. In addition, the 17-kDa protein encoded by the 9-27 gene was shown to be identical to the Leu-13 antigen. Leu-13 was previously identified as a 16-kDa interferon-inducible protein in leukocytes and endothelial cells and is a component of a multimeric complex involved in the transduction of antiproliferative and homotypic adhesion signals. These results suggest a novel level of cellular regulation by interferons involving a membrane protein, encoded by the interferon-inducible 9-27 gene, which associates with other proteins at the cell surface, forming a complex relaying growth inhibitory and aggregation signals.

Interferon-alpha induces the expression of the L-selectin homing receptor in human B lymphoid cells.

The L-selectin homing receptor expressed by lymphocytes mediates the initial attachment of these cells to high endothelial venules within peripheral lymph nodes. This adhesive interaction is required for the migration of B and T lymphocytes from the blood into peripheral lymph nodes. There is currently little information regarding the nature of the factors involved in the regulation of the synthesis and expression of L-selectin by lymphocytes. In this report, the immunomodulatory cytokine interferon-alpha (IFN-alpha) was shown to markedly upregulate the surface density of L-selectin in the established human B lymphoid Daudi cell line and in a subpopulation of tissue-derived human B lymphoid cells. Other cytokines such as IFN-gamma, tumor necrosis factor-alpha, interleukin (IL)-1 beta, IL-2, IL-4, IL-6, and low molecular weight B cell growth factor did not affect L-selectin surface expression in the model Daudi B cell line. Upregulation of L-selectin surface density in IFN-alpha-treated Daudi B cells correlated directly with an increase in L-selectin mRNA steady state levels and enhanced L-selectin-dependent binding to a carbohydrate-based ligand, phosphomonoester core polysaccharide. Regulation of L-selectin mRNA by IFN-alpha had characteristics similar to that of classical IFN-stimulated genes including rapid kinetics of induction, protein-synthesis-independent induction, and sensitivity to tyrosine-kinase inhibitors. IFN-alpha did not upregulate L-selectin mRNA levels or surface expression in an IFN-resistant Daudi subclone which exhibits a defect in the signal transduction pathway required for the transcriptional induction of IFN-stimulated genes. These data demonstrate a fundamental role for IFN-alpha in regulating L-selectin synthesis and expression in human B lymphoid cells and suggest a mechanism whereby this cytokine regulates the regional trafficking of B cells to peripheral lymph nodes.

IFN-alpha induces homotypic adhesion and Leu-13 expression in human B lymphoid cells.

IFN-alpha influences the recirculation and growth of normal and malignant B lymphocytes, although the mechanisms involved are not currently known. Lymphocyte recirculation is fundamentally dependent on cell-to-cell interactions that are mediated by cell surface adhesion molecules. In this report, we examined the relationship between the effect of IFN-alpha on cell-to-cell adhesion processes and induction of the Leu-13 cell surface protein in established human Daudi B lymphoid cell lines that are either sensitive or resistant to the antiproliferative activity of IFN-alpha. IFN-alpha directly triggered homotypic adhesion of IFN-sensitive Daudi B cells in a time- and dose-dependent manner. In contrast, IFN-alpha had no effect on the cell-to-cell adhesion of IFN-resistant Daudi B cells. The capacity of IFN-alpha to trigger homotypic aggregation correlated directly with the level of induction of the cell surface protein Leu-13 and could be potentiated by anti-Leu-13 mAb. Other cytokines also known to influence the proliferation, differentiation, or recirculation of B lymphocytes such as IFN-gamma, IL-2, IL-4, IL-6, TNF-alpha, and low molecular weight B cell growth factor did not induce either Leu-13 expression or homotypic aggregation of Daudi B cells. The adhesion pathway triggered by the IFN-inducible protein Leu-13 required metabolic energy and an intact cytoskeleton but was not dependent on: 1) new protein synthesis; 2) protein kinase C, protein kinase A, or tyrosine kinase activities; or 3) the function of known adhesion molecules including LFA-1, ICAM-1, CD44, or VLA-4. Taken together, these studies demonstrate a fundamental role for IFN-alpha and the IFN-inducible protein Leu-13 in regulating a novel homotypic adhesion pathway in B lymphocytes, and provide insight into the possible mechanisms by which IFN-alpha regulates biologic processes including recirculation.

Interferon-alpha alters spectrin organization in normal and leukemic human B lymphocytes.

Interferon-alpha (IFN-alpha) regulates the growth, differentiation, and recirculation of normal and malignant B lymphocytes. In this report we examine the effects of IFN-alpha on the distribution of the cytoskeletal protein spectrin in peripheral blood B lymphocytes from normal donors and patients diagnosed with chronic lymphocytic leukemia (CLL) and hairy cell leukemia (HCL). Exposure of normal and leukemic B cells to IFN-alpha in vitro was shown by immunofluorescence microscopy to cause a dose-dependent increase in the percentage of cells containing discrete focal accumulations of spectrin, ie, a single large aggregate or cap-like structure near the plasma membrane. Although the magnitude of this effect was variable among individual patient samples, in some experiments IFN-alpha induced a fourfold increase in the percentage of leukemic B cells exhibiting focal accumulations of spectrin. Spectrin reorganization induced by IFN-alpha was abrogated by the protein synthesis inhibitor cycloheximide. In addition, IFN-alpha increased the total cellular content of spectrin in B-CLL cells by approximately twofold to fourfold. Finally, a role for protein kinase C in mediating the effects of IFN-alpha on spectrin's organization is implicated by studies in which calphostin C inhibited the IFN-induced focal accumulation of spectrin. Taken together, these studies suggest that the immunomodulatory activities of IFN-alpha in normal and malignant B cells involve a change in the organization of the spectrin-based cytoskeleton.