Daclizumab reduces CD25 levels on T cells through monocyte-mediated trogocytosis.

Daclizumab is a humanized monoclonal antibody that prevents interleukin-2 (IL-2) binding to CD25, blocking IL-2 signaling by cells that require high-affinity IL-2 receptors to mediate IL-2 signaling. The phase 2a CHOICE study evaluating daclizumab as a treatment for multiple sclerosis (MS) included longitudinal analysis of activated T cell counts. Whereas an exposure-dependent relationship was observed between daclizumab and reductions in HLA-DR(+)-activated T cells, a similar relationship was not observed for reductions in CD25 levels. The objective of this report is to determine the mechanism by which daclizumab reduces CD25 levels on peripheral blood mononuclear cells (PBMCs) using cytometric techniques. Daclizumab reduced T cell CD25 levels through a mechanism that required the daclizumab-Fc domain interaction with Fc receptors (FcR) on monocytes, but not on natural killer (NK) cells, and was unrelated to internalization or cell killing. Activated CD4(+) T cells and FoxP3(+) Treg cells showed evidence of trogocytosis of the CD25 antigen in the presence of monocytes. A daclizumab variant that retained affinity for CD25 but lacked FcR binding did not induce trogocytosis and was significantly less potent as an inhibitor of IL-2-induced proliferation of PBMCs. In conclusion, Daclizumab-induced monocyte-mediated trogocytosis of CD25 from T cells appears to be an additional mechanism contributing to daclizumab inhibition of IL-2 signaling.

Control of apoptosis signaling by Apo2 ligand.

Apo2 ligand (Apo2L, also called TRAIL) is a member of the tumor necrosis factor (TNF) cytokine family. The closest homolog of Apo2L is CD95 (Fas/Apo1) ligand, to which it has 24% amino acid sequence identity. Similar to CD95L, Apo2L activates rapid apoptosis in many types of cancer cells; however, whereas CD95L mRNA expression is restricted mainly to activated T cells, natural killer cells, and immune-privileged sites, Apo2L mRNA occurs in a wide variety of tissues. Most normal cells appear to be resistant to Apo2L's cytotoxic action, suggesting the existence of mechanisms that can protect against apoptosis induction by Apo2L. The first receptor described for Apo2L, called death receptor 4 (DR4), contains a cytoplasmic "death domain"; DR4 transmits the apoptosis signal carried by Apo2L. We have identified three additional receptors that bind to Apo2L. One receptor, called DR5, contains a cytoplasmic death domain and signals apoptosis much like DR4. The DR4 and DR5 mRNAs are expressed in many normal tissues and tumor cell lines. The second receptor, designated decoy receptor 1 (DcR1), is a phospholipid-anchored cell-surface protein that lacks a cytoplasmic tail. The third receptor, called DcR2, is structurally similar to DR4 and DR5 but has a truncated cytoplasmic death domain and does not transmit a death signal. The mRNAs for DcR1 and DcR2 are expressed in multiple normal tissues but in few tumor cell lines. Transfection experiments indicate that DcR1 and DcR2 act as decoys that prevent Apo2L from inducing apoptosis through DR4 and DR5. These decoy receptors thus represent a novel mechanism for regulating sensitivity to a pro-apoptotic cytokine directly at the cell's surface. The preferential expression of these inhibitory receptors in normal tissues suggests that Apo2L may be useful as an anticancer agent that induces apoptosis in cancer cells while sparing normal cells.

Identification of a ligand for the death-domain-containing receptor Apo3.

The tumor necrosis factor (TNF) cytokine family regulates development and function of the immune system [1]. TNF is expressed primarily by activated lymphocytes and macrophages and induces gene transcription or apoptosis in target cells [2,3]. We have identified a novel relative of TNF that binds to the recently discovered, death-domain-containing receptor called Apo3 [4] (also known as DR3, WSL-1, TRAMP or LARD [5-9]). The Apo3 ligand (Apo3L) is a 249 amino-acid, type II transmembrane protein. The extracellular sequence of Apo3L shows highest identity to that of TNF. We detected Apo3L mRNA in many human tissues and mapped its encoding gene to chromosome 17p13, near the p53 tumor-suppressor gene. Soluble Apo3L induced apoptosis and nuclear factor kappaB (NF-kappaB) activation in human cell lines. Caspase inhibitors blocked apoptosis induction by Apo3L, as did a dominant-negative mutant of the cell death adaptor protein Fas-associated death domain protein (FADD/MORT1), which is critical for apoptosis induction by TNF [3]. Dominant-negative mutants of several factors that play a key role in NF-kappaB induction by TNF [10] inhibited NF-kappaB activation by Apo3L. Thus, Apo3L has overlapping signaling functions with TNF, but displays a much wider tissue distribution.

Control of TRAIL-induced apoptosis by a family of signaling and decoy receptors.

TRAIL (also called Apo2L) belongs to the tumor necrosis factor family, activates rapid apoptosis in tumor cells, and binds to the death-signaling receptor DR4. Two additional TRAIL receptors were identified. The receptor designated death receptor 5 (DR5) contained a cytoplasmic death domain and induced apoptosis much like DR4. The receptor designated decoy receptor 1 (DcR1) displayed properties of a glycophospholipid-anchored cell surface protein. DcR1 acted as a decoy receptor that inhibited TRAIL signaling. Thus, a cell surface mechanism exists for the regulation of cellular responsiveness to pro-apoptotic stimuli.

A novel receptor for Apo2L/TRAIL contains a truncated death domain.

Apo2 ligand (Apo2L [1], also called TRAIL for tumor necrosis factor (TNF)-related apoptosis-inducing ligand [2]) belongs to the TNF family and activates apoptosis in tumor cells. Three closely related receptors bind Apo2L: DR4 and DR5, which contain cytoplasmic death domains and signal apoptosis, and DcR1, a decoy receptor that lacks a cytoplasmic tail and inhibits Apo2L function [3-5]. By cross-hybridization with DcR1, we have identified a fourth Apo2L receptor, which contains a cytoplasmic region with a truncated death domain. We subsequently named this protein decoy receptor 2 (DcR2). The DcR2 gene mapped to human chromosome 8p21, as did the genes encoding DR4, DR5 and DcR1. A single DcR2 mRNA transcript showed a unique expression pattern in human tissues and was particularly abundant in fetal liver and adult testis. Upon overexpression, DcR2 did not activate apoptosis or nuclear factor-kappaB; however, it substantially reduced cellular sensitivity to Apo2L-induced apoptosis. These results suggest that DcR2 functions as an inhibitory Apo2L receptor.

Apo-3, a new member of the tumor necrosis factor receptor family, contains a death domain and activates apoptosis and NF-kappa B.

BACKGROUND: Two receptors that contain the so-called "death domain' have been described to date: tumor necrosis factor receptor 1 (TNFR1) and Fas/Apo-1 (CD95); both belong to the TNFR gene family. The death domain of TNFR1 mediates the activation of programmed cell death (apoptosis) and of the transcription factor NF-kappa B, whereas the death domain of CD95 only appears to activate apoptosis. RESULTS: We have identified an additional member of the TNFR family, which we have named Apo-3. Apo-3 is a transmembrane protein of approximately 47 kDa that has similarity of members of the TNFR family in its extracellular, cysteine-rich domains. In addition, Apo-3 resembles TNFR1 and CD95 in that it contains a cytoplasmic death domain. The Apo-3 gene mapped to human chromosome 1p36.3, and Apo-3 mRNA was detected in several human tissues, including spleen, thymus, peripheral blood lymphocytes, small intestine and colon. Ectopic expression of Apo-3 in HEK293 or HeLa cells induced marked apoptosis. CrmA, a poxvirus inhibitor of Ced-3-like proteases which blocks death signaling by TNFR1 and CD95, inhibited Apo-3-induced apoptosis. Ectopic expression of Apo-3 also induced the activation of NF-kappa B. Apo-3 did not specifically bind to the Apo-2 ligand, suggesting the existence of a distinct ligand for Apo-3. CONCLUSIONS: These results identify Apo-3 as a third member of the TNFR family that activates apoptosis, and suggest that Apo-3, TNFR1 and CD95 engage a common apoptotic cell-death machinery. Apo-3 resembles TNFR1 because it can stimulate NF-kappa B activity and regulate apoptosis. Apo-3 mRNA is expressed in various tissues, consistent with the possibility that this receptor may regulate multiple signaling functions.

Induction of apoptosis by the anti-tubulin drug colcemid: relationship of mitotic checkpoint control to the induction of apoptosis in HeLa S3 cells.

We have studied the relationship between apoptosis and drug-induced cell cycle perturbation in HeLa S3 cells when treated with the anti-tubulin drug colcemid. We found that at least two distinct mechanisms contributed to colcemid cytotoxicity and apoptosis. Continuous exposure to concentrations of colcemid sufficient to block cells at the mitotic checkpoint led to the appearance of apoptotic cells approximately one cell cycle after their initial accumulation in mitosis. Continuous exposure to concentrations sufficient to delay mitotic progression but insufficient to cause mitotic arrest, or pulse exposure to concentrations of colcemid sufficient to induce mitotic block, led to the generation of multipolar mitoses and genetically deficient hypodiploid daughter cells which underwent apoptosis while in interphase. The fact that aberrant spindle function delayed but did not block cells at the mitotic checkpoint indicates that the mitotic checkpoint senses the presence or absence of the spindle but not spindle abnormalities. In both mitotic and interphase cells, colcemid-induced apoptosis occurred after a period of cell cycle stasis during which cells failed to complete an initiated cell cycle. These results are discussed with reference to understanding the relationship between apoptosis and the regulation of cell cycle progression.

Harmonic analysis of intravascular pressure response as an index of arterial wall mechanical properties.

The ability to routinely assess mechanical properties of large blood vessels, like the aorta, before an aneurysm or rupture occurs, could benefit diagnostic and therapeutic procedures and save lives. In this study, images of the wall area and intravascular pressure (IP) responses of in vitro rat aorta were recorded during swept frequency pressure input (2-200 Hz; +/- 10 mm Hg) superimposed on mean pressures from 20 to 160 mm Hg. Data analysis included Fast Fourier transform (FFT) of input and responses. Wall and IP responses were underdamped with respective resonance frequencies (Wn) that varied as a function of mean input pressure and the nonlinear nature of wall elasticity. Results indicated closely coupled wall and IP responses and suggested that the IP response may be an adequate index of wall elasticity without need of a direct measure of wall displacement. We considered results to be a key step towards development of a clinical tool which would facilitate analysis of mechanical properties of in vivo conducting vessels.

Synchronizing blood vessel response CCD imagery to swept frequency dynamic pressure signals.

Harmonic analysis of the pressure and wall responses of a blood vessel exposed to a dynamic pressure input signal required the development of a software application which could properly synchronize the data gathered by two separate microcomputers. In order to accomplish this task, the Pressure-Image Editor was developed. The first computer is used to generate a swept frequency sinusoidal dynamic pressure input signal while at the same time monitoring the resulting response pressures. The second computer is used to record the physical (visual) response of the artery to the pressure signal via a high speed CCD camera and video digitizer. Using the Pressure-Image Editor, 256 animated images along with 65,536 pressure points can be combined and synchronized based on the camera frame rate, input trigger frequency, and any internal timing delays. The Pressure-Image Editor is a object-oriented application written in C++ and includes a window based graphical user interface.

Transforming growth factor-beta induces platelet-derived growth factor (PDGF) messenger RNA and PDGF secretion while inhibiting growth in normal human mammary epithelial cells.

Platelet-derived growth factor (PDGF) is a potent mitogen in human serum which specifically stimulates the proliferation of mesenchymal cells. We have now examined normal human mammary epithelial cells (HMEC) derived from reduction mammaplasties and grown in a serum-free defined medium. Medium conditioned by HMEC contained a PDGF-like activity that competed with [125I]PDGF for binding to PDGF receptors in normal human fibroblasts. When conditioned media were incubated with antiserum specific for either PDGF-A or PDGF-B, only PDGF-A antiserum was capable of inhibiting binding of conditioned media to PDGF receptors. Using an RNase protection assay, mRNA from normal HMEC was probed for both the PDGF-A and PDGF-B chains. Little or no PDGF-B was found in HMEC strains, while a strong signal was seen with the PDGF-A probe. When HMEC were grown in the presence of transforming growth factor-beta (TGF beta) for 48 h, inhibition of growth was observed in association with a 20- to 40-fold stimulation of PDGF-B mRNA and a 2-fold stimulation of PDGF-A mRNA. This mRNA induction was extremely rapid (within 1 h), and secreted PDGF activity was induced 2- to 3-fold. Two other HMEC growth inhibitors and differentiating agents, sodium butyrate and phorbol ester 12-O-tetradecanoylphorbol-13-acetate, had no effect on PDGF mRNA regulation. The current study suggests that PDGF gene induction is an extremely rapid and specific indicator of TGF beta function regardless of whether TGF beta is acting in a growth stimulatory or inhibitory manner. Any role of PDGF-B in TGF beta modulation of differentiation of normal or malignant mammary gland remains to be determined.

Expression of the transforming growth factor-alpha/epidermal growth factor receptor pathway in normal human breast epithelial cells.

To better understand the possible roles and interactions of transforming growth factor-alpha (TGF alpha) and its receptor, the epidermal growth factor (EGF) receptor in human breast epithelium, we have studied the expression of TGF alpha and the EGF receptor in a series of normal human mammary epithelial cells derived from reduction mammoplasty before in vitro propagation, during short term proliferation in vitro, and after immortalization. Increased TGF alpha mRNA expression coincided with conversion of the cells to a proliferative state in vitro. After establishment, propagation, and proliferation in vitro, the cells expressed high levels of both TGF alpha and EGF receptor mRNAs. Addition of diverse growth inhibitory agents, including 12-O-tetradecanoylphorbol-13-acetate (TPA), TGF beta, and sodium butyrate, to one of these rapidly proliferating cell populations (no. 184) failed to reduce the expression of either TGF alpha or the EGF receptor. Likewise, cessation of growth associated with both senescence and confluence of the 184 cells did not result in reduced expression. However, regulation of TGF alpha mRNA could be demonstrated by withdrawal of EGF from the medium or by antibody-mediated blockade of the EGF receptor in 184 cells. Antibody-mediated EGF receptor blockade also results in inhibition of growth and [3H]thymidine labeling. An autoregulatory autocrine loop appears operant in proliferating breast epithelial cells. Both growth and levels of TGF alpha mRNA expression are controlled by binding of ligand to the EGF receptor. These studies suggest a role for the TGF alpha/EGF receptor pathway in normal breast cell physiology.

Differential scanning calorimetric study of the thermotropic phase behavior of a polymerizable, tubule-forming lipid.

A comparative study of the polymorphism exhibited by the polymerizable, tubule-forming phospholipid 1,2-bis(10,12-tricosadiynoyl)-sn-glycero-3- phosphocholine (DC23PC) and its saturated analog 1,2-ditricosanoyl-sn-glycero-3-phosphocholine (DTPC) in aqueous suspension is reported. Differential scanning calorimetry (DSC), as well as freeze-fracture electron microscopy and Raman spectroscopy, have been used to study the influence on phase behavior of rigid diacetylene groups in the fatty acyl chains of a phosphatidylcholine. DTPC large multilamellar vesicle (MLV) and small unilamellar vesicle (SUV) suspensions were found to retain liposome morphology after chain crystallization had occurred. In marked contrast, diacetylenic DC23PC suspensions do not maintain liposomal morphology in converting to the low temperature phase. Large MLVs of DC23PC with outer diameters in excess of 1 micron convert to a gel phase with cylindrical or tubular morphology at 38 degrees C, just a few degrees below the lipid's chain melting temperature (TM(H), i.e. temperature of an endothermic event observed during a heating scan) of 43.1 degrees C. Unlike the large MLVs, small MLVs or SUVs of DC23PC, with diameters of 0.4 +/- 0.3 micron and 0.04 +/- 0.02 micron, respectively, exhibit metastability in the liquid-crystalline state for several tens of degrees below the chain melting temperature prior to converting to a gel phase which, by electron microscopy, manifests itself as extended multilamellar sheets. Raman data collected at TM(H) -40 degrees C demonstrate that the gel state formed by DC23PC is very highly ordered relative to that of DTPC, suggesting that special chain packing requirements are responsible for the novel phase behavior of DC23PC.

A homogeneous immunoassay for the mycotoxin T-2 utilizing liposomes, monoclonal antibodies, and complement.

The trichothecene mycotoxin T-2 is a fungal metabolite known to contaminate agricultural products and cause intoxication of humans and animals. We have developed a homogeneous competition inhibition assay for T-2 mycotoxin based on complement-mediated lysis of liposomes. The T-2 mycotoxin was converted to an acid chloride derivative, subsequently coupled to the amino group of phosphatidylethanolamine, and incorporated with the phospholipid into unilamellar liposomes. Carboxyfluorescein, which is self-quenched at high concentrations, was entrapped in the liposomes as a release marker. We used a monoclonal IgG1 antibody specific for T-2 mycotoxin and a polyclonal anti-mouse Ig as a secondary antibody since the anti-T-2 IgG1 does not activate complement. In the absence of free T-2, the liposomes were lysed within 30 min after the addition of complement, releasing carboxyfluorescein into the surrounding buffer. In the presence of free T-2 toxin, the binding of antibodies to the liposomes was reduced, causing a corresponding decrease in lysis. This assay proved to be sensitive to T-2 toxin levels as low as 2 ng, which is 10-fold more sensitive than the present enzyme immunoassay using the same antibodies.

Encapsulation of hemoglobin in phospholipid vesicles.

Hemoglobin has been encapsulated in phospholipid vesicles by extrusion of hemoglobin/lipid mixtures through polycarbonate membranes. This technique avoids the use of organic solvents, sonication, and detergents which have proven deleterious to hemoglobin. The vesicles are homogeneous, with a mean size of 2400 A as determined by photon correlation spectroscopy. The encapsulated hemoglobin binds oxygen reversibly and the vesicles are impermeable to ionic compounds. Hemoglobin encapsulated in egg phosphatidylcholine vesicles converts to methemoglobin within 2 days at 4 degrees C. By contrast, when a mixture of dimyristoyl phosphatidylcholine, cholesterol and dicetyl phosphate is used there is no acceleration in methemoglobin formation, and the preparation is stable for at least 14 days at 4 degrees C.

Changes in size and shape of liposomes undergoing chain melting transitions as studied by optical microscopy.

Changes in the shape and size of dipalmitoylphosphatidylcholine liposomes at the phase transition at 41.5 degrees C have been monitored by light microscopy. All liposomes change size or shape at the transition and those with simple topologies such as spheres and cylinders can be readily measured. The surface area of these is some 24% greater above the transition than below. This surface area change is virtually identical to that predicted by crystallographic measurements on this system. Also, the rate of transition from one state to another is seen to proceed more rapidly in the smaller liposomes. Optical microscopic observation provides a rapid simple method for monitoring the dependence of the lipid bilayer area on temperature.

Photon correlation spectroscopy study on the stability of small unilamellar DPPC vesicles.

The growth in size of dipalmitoylphosphatidylcholine small unilamellar vesicles (SUV) below Tm has been studied by photon correlation spectroscopy and differential scanning calorimetry. We see an initial fast rise of the hydrodynamic diameter of the vesicles followed by a slower increase. We assign the slow component of the size change to fusion of SUV. The order of the kinetics appears to be higher than first order. The estimated half lifetime of the fusion is approximately 67 h. The diameters for the fast and slow processes at t = O are 756 and 256 A, respectively, while as t leads to infinity the diameters increase to 1,570 and 733 A, respectively.

Kinetic and thermodynamic studies of the fusion of small unilamellar phospholipid vesicles.

Small phospholipid vesicles, prepared so as to minimize impurities, fuse relatively slowly resulting in the time-dependent development of a characteristic endotherm in differential scanning calorimetry and corresponding changes in the Raman spectrum. The stability of small vesicles towards fusion increases with increasing acyl chain length for the series C-14 through 18. Within the protocols of these experiments, the fusion rate remains unchanged whether the vesicles are held at 10 degrees C below Tm or at Tm itself. We have determined enthalpies of transition for small vesicles and fusion product for C-14 through C-18. In each case delta H for small vesicles is lower than that of the corresponding multilamellar vesicles, while the fusion product delta H is intermediate between small and multilamellar vesicles. The apparent lack of concensus in the literature as to the nature of the fusion process is ascribed to the variety of protocols used as well as the presence or absence of fusion-inducing impurities.

Resonance Raman scattering from uteroferrin, the purple glycoprotein of the porcine uterus.

Uteroferrin, a purple-colored, iron-containing glycoprotein, purified from the uterine fluid of progesterone-stimulated pigs, owes its natural purple color to a broad absorption band centered at 545 nm. Laser excitation within the visible absorption band of uteroferrin results in an intense resonance Raman spectrum which bears a striking resemblance to that reported for Fe(III)-transferrin, the iron transport protein of serum. Excitation profiles for the four resonance-enhanced bands of uteroferrin were obtained from 4579 A to 6741 A, using lines from Ar+ and Kr+ lasers. Each of the profiles have maxima near 545 nm. The spectral similarities of uteroferrin and Fe(III)-transferrin lead to the belief that the Fe(III) binding sites of the two proteins must be, at least in some respects, quite similar. In particular, it is concluded that, as in Fe(III)-transferrin, the metal binding site of uteroferrin contains a tyrosine ligand and that the visible absorption spectrum of uteroferrin results from a phenolate to Fe(III) charge transfer.