Caspase 8 activity in membrane blebs after anti-Fas ligation.

Previous studies of thymocyte apoptosis using a series of cell-permeable fluorogenic peptide substrates showed that Fas cross-linking triggered a caspase cascade in which cleavage of the IETDase (caspase 8-selective) substrate was the earliest caspase activity measured by flow cytometry. This result was expected in light of the abundant evidence for caspase 8 activation as an initiating event in the Fas death pathway. However, when apoptosis was induced by anti-Fas in CTL and the caspase cascade examined by this approach, IETDase activation followed increases in LEHDase, YVHDase, and VEIDase activities (selective for caspases 9, 1, and 6, respectively). When examined by confocal microscopy, anti-Fas-treated CTL showed the early appearance of IETDase-containing plasma membrane vesicles and their release from the CTL surface, followed by activation of other caspase activities in the cell interior. Since these vesicles were not included in the flow cytometry analysis, the early IETDase activity had been underestimated. In contrast to anti-Fas, induction of apoptosis in these CTL by IL-2 withdrawal resulted in early IETDase activity in the cytoplasm, with no plasma membrane vesiculation. Thus, anti-Fas-induced initiation of caspase activity at the plasma membrane may in some cells result in local proteolysis of submembrane proteins, leading to generation of membrane vesicles that are highly enriched in active caspase 8.

Assessment of caspase activities in intact apoptotic thymocytes using cell-permeable fluorogenic caspase substrates.

To detect caspase activities in intact apoptotic cells at the single cell level, cell-permeable fluorogenic caspase substrates were synthesized incorporating the optimal peptide recognition motifs for caspases 1, 3/7, 6, 8, and 9. Caspase activities were then assessed at various times after in vitro treatment of mouse thymocytes with dexamethasone or anti-Fas antibody. Dexamethasone induced the following order of appearance of caspase activities as judged by flow cytometry: LEHDase, WEHDase, VEIDase, IETDase, and DEVDase. Since the relative order of caspases 3 (DEVDase) and 6 (VEIDase) in the cascade has been controversial, this caspase activation order was reexamined using confocal microscopy. The VEIDase activity appeared before DEVDase in every apoptotic cell treated with dexamethasone. In contrast, anti-Fas stimulation altered this sequence: IETDase was the first measurable caspase activity and DEVDase preceded VEIDase. In an attempt to determine the intracellular target of the potent antiapoptotic agent carbobenzoxy-valyl-alanyl-aspartyl(beta-methyl ester)-fluoromethyl ketone (Z-VAD[OMe]-FMK), we examined its ability to inhibit previously activated intracellular caspases. However, no significant reductions of these activities were observed. These fluorogenic caspase substrates allow direct observation of the caspase cascade in intact apoptotic cells, showing that the order of downstream caspase activation is dependent on the apoptotic stimulus.

A nanosecond fluorescence study of the simultaneous influx of Ca2+ and Cd2+ into liposomes.

Nanosecond fluorescence decay characteristics of the calcium-binding probe Quin2 and two of its cation complexes were examined by time-resolved fluorescence spectroscopy. Binding of Ca2+ and Cd2+ resulted in fluorescence lifetime enhancements as compared to that of free Quin2 ('tau' = 0.9 ns). The Quin2-Ca2+ complex displays a monoexponential decay of tau = 7.4 ns, while the cadmium complex gives an average decay time of ca. 4 ns. Lifetime measurements made on heterogeneous cationic solutions demonstrate that decay times for individual complexes can be retrieved. Time-resolved measurements were used to monitor the kinetics of ionomycin-mediated calcium and cadmium transport across artificial membranes. Fluorescence decays, collected on the time-scale of second, were sufficient to measure individual ion fluxes or those of mixtures into liposomes. The combination of steady-state and time-resolved fluorescence techniques offers the unique advantage of simultaneously detecting other cations in the presence of calcium.

Tumor cell recognition by lymphocytes: is the MHC always essential?

Phenotypic and functional analyses of tumor-infiltrating lymphocytes (TILs) used in clinical trials revealed that cells other than CTLs can have antitumor efficacy. This observation led us to search for mechanisms for tumor recognition by lymphocytes that utilize alternatives to surface structures of tumor cells, for example, MHC antigen complexes; the latter are generally believed to be the immunogenic platforms for CTLs. Therefore, as a possible source of immunostimulatory activity, we compared the ability of plasma membrane components of tumor cell lines with first secreted tumor cell components and then intracellular tumor components to act as mitogenic sources for human TIL lines. Surprisingly, the latter was found to be most potent, particularly Oncoimmunin-L, which is a 45-kDa protein with sequence similarity to members of the serpin family of proteins. This protein, which has at least a 31% sequence identity to human leukocyte elastase inhibitor and stimulates [3H]-thymidine incorporation into the DNA of human TILs, may be found in the cytosol of many tumor cells. Taken together with our earlier work in which a 36-kDa protein, also of tumor cytosolic origin, was shown to induce differentiation of myeloid cells, we propose soluble factors derived from tumor cells as a pathophysiological source of tumor immunogenicity. Moreover, detailed biochemical and biophysical characterization of tumor cell-immunocyte interactions will define the tumor immunoenvironment.

Characterization of fluorescence quenching in bifluorophoric protease substrates.

NorFES is a relatively rigid, bent undecapeptide which contains an amino acid sequence that is recognized by the serine protease elastase (AspAlaIleProNle downward arrow SerIleProLysGlyTyr ( downward arrow indicates the primary cleavage site)). Covalent attachment of a fluorophore on each side of NorFES's elastase cleavage site enables one to use a change of fluorescence intensity as a measure of enzymatic activity. In this study two bichromophoric NorFES derivatives, D-NorFES-A and D-NorFES-D, were prepared in which D (donor) was tetramethylrhodamine and A (acceptor) was rhodamine-X, two chromophores with characteristics suitable for energy transfer. Absorption and fluorescence spectra were obtained with both the intact and cleaved homodoubly, heterodoubly and singly labeled derivatives. It was found that both the homo and hetero doubly-labeled derivatives form ground-state complexes which exhibit exciton bands. The hetero labeled derivative exhibits little or no resonance energy transfer. Spectral measurements were also done in urea, which partially disrupts ground-state dimers.

Steady-state and time-resolved fluorescence measurements for studying molecular interactions: interaction of a calcium-binding probe with proteins.

The binding of 2-[(2-bis-[carboxymethyl]amino-5-methylphenoxy)-methyl] 6-methoxy-8-bis[carboxymethyl] aminoquinoline, the fluorescent calcium probe Quin2, to serum albumin and several other proteins has been investigated. Changes in fluorescence emission spectra and fluorescence anisotropy revealed interactions between Quin2 and several proteins including human serum albumin, bovine serum albumin, aldolase, phosphoglucose isomerase, glyceraldehyde-3-phosphate dehydrogenase, and alkaline phosphatase. Protein-probe interactions were inhibited by the presence of calcium. Binding was also measured by resonance energy transfer and gel permeation chromatography. Equilibrium binding constants for Quin2 were quantitated by the application of the recently-developed "SPECTRABIND' program to spectroscopic data (D. Toptygin and L. Brand, Anal. Biochem., 224 (1995) 330-338). Binding of Quin2 to human serum albumin is discussed in terms of the published X-ray crystal structure of human serum albumin (X.M. He and D.C. Carter, Nature, 358 (1992) 209-215).

Profluorescent protease substrates: intramolecular dimers described by the exciton model.

Xanthene dyes are known to form dimers with spectral characteristics that have been interpreted in terms of exciton theory. A unique aspect of H-type dimers is the fluorescence quenching that accompanies their formation. Using the principles of exciton theory as a guide, a series of protease substrates was synthesized with a xanthene dye on each side of the cleavage site. To bring the attached dyes into spatial proximity to form a dimer, the molecular design included structure determinant regions in the amino acid sequence. In addition, chromophores were chosen such that changes in absorption spectra indicative of exciton splitting were anticipated. Cleavage of the peptides by a protease resulted in disruption of the dimers and indeed significant absorption spectral changes were observed. Furthermore, substrate cleavage was accompanied by at least an order of magnitude increase in fluorescence intensity. This has allowed determination of intracellular elastase activity using a fluorescence microscope equipped with standard optics.

A serpin from human tumor cells with direct lymphoid immunomodulatory activity: mitogenic stimulation of human tumor-infiltrating lymphocytes.

A serum-free supernatant from an epidermal carcinoma cell line has previously been shown to contain mitogenic activity for human tumor infiltrating lymphocytes in culture [1]. From this conditioned medium we have now purified to homogeneity, as determined by SDS-PAGE analysis, a ca. 45 kDa protein which stimulates [3H]thymidine incorporation into the DNA of these human T-lymphocytes. Amino acid composition data and immunoreactivity of the purified protein as well as sequence analyses of 7 tryptic fragments obtained therefrom suggest a strong similarity with human monocyte/neutrophil elastase inhibitor, which is a member of the serine protease inhibitor (serpin) superfamily. We have previously identified and purified from the same conditioned medium a 36 kDa protein with myeloid immunomodulatory activity [2]. Taken together, these two reports support the role of tumor-derived soluble factors in tumor immunosurveillance.

Mitogenic stimulation of human lymphocytes mediated by a cell surface elastase.

A ca. 45-kDa protein which was recently identified and purified to homogeneity from a solid tumor cell line as a T-cell mitogen was found to have significant sequence similarities with human monocyte/neutrophil elastase inhibitor (EI) [1]. Since EI is a known substrate for elastase, a determination of whether a cell surface expressed elastase-like molecule might be the binding protein for this 45-kDa factor and mediate mitogenic signal transduction was undertaken. First, the surface of tumor infiltrating lymphocytes, TIL 660, the indicator cell line used for the purification of this mitogen, was shown to stain positively with an anti-elastase antibody using flow cytofluorometry for quantitation. Then, after observing an inverse correlation between cell surface staining and the proliferative status of the TILs, behavior which might be expected of a growth factor receptor upon activation, mitogenic signal transduction was attempted through the elastase-like molecules of the lymphocytes' plasma membrane with the anti-elastase antibody in the role of mitogen. A greater than 4-fold mitogenic stimulation was observed when this antibody was covalently linked to latex beads; in contrast, addition of the soluble form of the same antibody did not result in any increase in [3H]thymidine incorporation into the cells' DNA. Hence, these data support induced clustering of an elastase-like molecule on the lymphocyte surface as a mediator of mitogenesis and suggest that the binding protein for mitogenic signal transduction induced by the 45-kDa protein, a member of the serine protease inhibitor (serpin) superfamily of proteins, is a molecule with structure similar to a serine protease.

Bone marrow activation by immobilized antibodies against tumor cells and immunocytes as a potential cancer immunotherapy.

This study was aimed at combining the limited expression pattern of the type 1 mucin glycoproteins on tumors derived from glandular epithelia with the presence of integrin adhesion receptors on hematopoietic cells in order to develop an immunotherapy against certain types of carcinomas. To this end, monoclonal antibodies that recognize molecules on the surfaces of either tumor cells or immunocytes were immobilized on latex beads; proliferation of bone marrow cells, representing a source of preterminally differentiated immunocytes with potential antitumor activity, was measured and compared with that of mature lymphocytes in the presence of beads and irradiated tumor cells. It was found that only beads carrying antibodies against both mucins and leukocyte integrins were capable of inducing proliferation of bone marrow cells while none specifically stimulated mature lymphocytes. A proposal is put forth for the development of tumor-induced proliferation of bone marrow cells as a potential effective immunotherapy for some forms of cancer.

Upregulation of the surface expression of two integrins and induction of chemotactic activity in a human leukemic cell line by Oncoimmunin-M.

Previously, it was shown that Oncoimmunin-M (OI-M), a recently identified tumor cell-derived 36 kDa protein, is able both to inhibit the proliferation of the human promyelocytic leukemic cell line HL-60 while maintaining viability in culture and to induce a bimodal distribution of CD11b, the alpha chain of the integrin MAC-1, on the cell surface (Packard, B.Z. and Komoriya, A. (1993) J. Biol. Chem. 268, 6356-6363). Now, data which reveal that exposure of HL-60 cells to this factor also brings about an increase in the mean level of surface expression of CD11c, the alpha chain of another leukocyte integrin (p150,95), but leaves CD11a, the alpha chain of the third leukointegrin (LFA-1), virtually unchanged (< 10%) are presented. Comparison of motility studies of OI-M-treated HL-60 bulk populations with control bulk populations demonstrates coinduction of CD11b and CD11c surface upregulation with chemotactic responsiveness to a gradient of the chemoattractant human C5a. Separation of motile from nonmotile cell subpopulations after exposure to C5a further reveals that individual cells which respond to this chemoattractant express increased levels of both CD11b and CD11c relative to unresponsive cells. These data correlate the upregulation of leukointegrins MAC-1 and p150,95 by a tumor cell-derived protein on a preterminally differentiated myeloid cell with chemotactic responsiveness to human C5a.

A role for intracellular components of target cells in activation of the immunosurveillance network.

The role of cells of lymphoid and myeloid origin as effectors in the immunosurveillance network is based upon their recognition of surface structures on target cells. The work described here, however, has focused on an alternative class of target molecules, that is, soluble intracellular factors, as potential immunogens. Results from this study, which was aimed at a biochemical definition of the tumor immunoenvironment, demonstrate that the soluble intracellular contents of a tumor cell line are significantly more effective than the extracellular medium conditioned by the same cells in both stimulating the growth of a human T-lymphocyte line and inducing differentiation markers in a human myeloid leukemic cell line. A proposal is made for a restructuring of the way in which the immunosurveillance network is considered. Specifically, it is suggested that the soluble intracellular components of tumor cells may serve as immunogens in the immunosurveillance network. It is further proffered that an understanding of the physical and chemical states of molecules which under pathologic conditions become exposed to effector components of the immunosurveillance network will give rise to new immunotherapeutic venues.

A 36-kilodalton tumor-derived factor with myeloid immunomodulatory activity.

The conditioned medium from the epidermal carcinoma cell line A431 is shown to inhibit the growth of three human myeloid leukemic cell lines. We have purified to homogeneity from this conditioned medium a 36-kDa protein, as measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, which inhibits [3H]thymidine incorporation into the DNA and induces cell surface expression of CD11b, the alpha chain of the adhesion receptor MAC-1, on the human promyelocytic leukemic cell line HL-60. This factor was purified by sequential anion exchange, hydrophobic interaction, and gel permeation chromatography. Amino acid sequence analysis of two tryptic fragments of the purified material showed greater than 95% homology with sequences 179-194 and 319-328 of the M chain of human L-lactate dehydrogenase. Although the tetrameric L-lactate dehydrogenase alone exhibits no activities associated with the purified 36-kDa protein, brief acid treatment which has been shown to yield predominantly monomeric lactate dehydrogenase-M1 results in about 50% of the maximal antiproliferative activity of that induced by this factor. The role of soluble factors of tumor origin in modulating myeloid function as part of an immunosurveillance network is discussed.