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Biomedical subjects

P Henkart

Publications and source records attributed to P Henkart.

At least 19 recordsLinked to original sources
PubMed1993

Hydrolysis of myelin basic protein in myelin membranes by granzymes of large granular lymphocytes.

Immune-mediated demyelination initially manifests as a separation of myelin lamellae followed by loss of myelin proteins and eventual loss of myelin membranes. Myelin basic protein, one of the major structural proteins of myelin, is highly vulnerable to various proteases derived from diverse cell types. Killer lymphocytes such as CTL, in addition to other immune effectors, have been implicated in inflammatory demyelination. In addition to a pore forming peptide, the granules of CTL and large granular lymphocytes (LGL) contain a number of serine esterases collectively called as granzymes. We studied the effect of these granule enzymes on myelin and found that LGL granule-extracts (LGL-g) hydrolyzed MBP in myelin membranes. LGL-g also cleaved purified MBP at neutral pH in a Ca2+ independent manner and this hydrolysis was inhibited by serine esterase inhibitors. In addition, absorption of LGL-g with antigranzyme A significantly reduced MBP hydrolysis. These findings implicated the granzymes in LGL-g, especially granzyme A, as the agent causing MBP degradation. Inasmuch as activation of CTL in the vicinity of myelinated axons can lead to granule exocytosis, hydrolysis of MBP by granzymes may be a significant event in myelin destruction.

Cell Membrane↗
PubMed1989

Inhibition of cytotoxic T cell lysis by anti-CD8 monoclonal antibodies: studies with CD3-targeted cytolysis of nominal antigen-negative targets.

The function of the CD8 molecule in lympholysis mediated by cytotoxic T cells was investigated by examining possible contributions of ligands on the target cell to the inhibition of lysis observed with CD8-specific mAb. In order to evaluate a variety of target cells, including those not expressing the nominal Ag (NA) for which the CTL was specific, lysis was effected by cross-linking the CTL and the target cells with anti-CD3 mAb. Such CD3 redirected cytotoxicity was demonstrated to be inhibited by anti-CD8 mAb when low anti-CD3 mAb concentrations were used. The possibility that inhibition by anti-CD8 mAb resulted for competition for the FcR between the anti-CD3 mAb and anti-CD8 mAb was eliminated by targeting TNP-modified cells with an antibody heteroconjugate prepared from Fab fragments of anti-CD3 and anti-DNP antibodies. Inhibition of the lysis of target cells not expressing NA including those deficient in class I expression, demonstrated that neither NA nor class I expression was required for anti-CD8 mAb inhibition. Whether the anti-CD8 mAb inhibition required CD8 Ag interaction with any ligand on the target cell was further investigated by measuring exocytosis of enzyme granule from CTL activated with CD3-coated poly-styrene beads. CD8-specific mAb inhibited such CTL activation in this target cell-free system. A CD8(+), MHC class II-specific CTL clone, was used to show differential inhibition by anti-CD8 mAb, depending on the target cell, therefore providing evidence that anti-CD8 mAb binding does not generate an absolute off signal. These data are consistent with the hypothesis that anti-CD8 mAb affect the lytic process independent of the recognition of a ligand on the target cell by CD8.

Antibodies, Monoclonal↗
PubMed1988

IL-2-dependent expansion of CD3+ large granular lymphocytes expressing T cell receptor-gamma delta. Evidence for a functional receptor by anti-CD3 activation of cytolysis.

The nature and function of the TCR on PBL of a patient with a chronic CD3+ large granular (LGL) proliferation was studied. Fresh peripheral blood from this individual was comprised of 80% lymphocytes, 65 to 75% of which were CD3+, CD8+, Leu-7+ LGL. Of these LGL, 72% initially expressed the TCR-alpha beta heterodimer, whereas 21% did not. Cytotoxicity directed against MHC-unrestricted targets was minimal. After several days of exposure to rIL-2, cytotoxic activity was greatly enhanced, correlating with a disappearance of CD3+ cells expressing the alpha beta heterodimer. Twelve days after rIL-2 exposure, the LGL expressed only TCR-gamma delta heterodimer in association with CD3 and alpha beta heterodimer expression could no longer be detected. The TCR/CD3 complex on these cells was demonstrated to be functional as anti-CD3 elicited an increase in cytoplasmic free calcium concentration, stimulated cytolytic activity, and stimulated granule enzyme secretion from the LGL.

Antigens, Differentiation, T-Lymphocyte↗
PubMed1987

Inhibition of NK and ADCC activity by antibodies against purified cytoplasmic granules from rat LGL tumors.

Highly purified preparations of cytoplasmic granules from transplantable rat large granular lymphocyte (LGL) tumor lines (rat natural killer (RNK) tumors) were used to immunize rabbits. Antibodies from these animals gave two precipitin lines with granule extracts in Ouchterlony experiments. They reacted with at least four different bands on nitrocellulose blots of SDS gels of LGL granule proteins. By immunofluorescence, specifically adsorbed antigranule antibodies did not recognize LGL or T cell surface antigens but reacted with the cytoplasmic granules in permeabilized RNK tumor cells as well as with normal rat LGL. These same antisera showed little or no reactivity with a panel of other cells, including peripheral blood T cells, thymocytes, macrophages, and EL-4 tumor cells. F(ab')2 preparations of these antigranule antibodies completely blocked granule-mediated lysis of both SRBC and nucleated targets, while control F(ab')2 preparations from rabbits immunized with EL-4 granules or TNP-KLH showed no significant inhibition of this cytolytic activity at the same antibody concentration. Anti-granule F(ab')2 preparations specifically inhibited (greater than 75%) rat natural killer (NK) and antibody-dependent cellular cytotoxicity (ADCC) activities in a dose-dependent manner but did not effect cytotoxic T cell activity. Pretreatment of either effectors or targets by these antibodies had no effect. Anti-granule F(ab')2 preparations, at concentrations showing strong inhibition of lysis, did not inhibit the binding of LGL to YAC-1 or Ab-coated P815 targets. These results demonstrate that a granule component(s) is necessary for the lytic activity of LGL in both NK and ADCC and provide the first direct evidence that a secretory event involving these granules is part of the lytic process.

Animals↗
PubMed1987

Secretory processes in lymphocyte function.

The secretion of immunoglobulin by plasma cells has been considered a classical example of the "non-regulated" pathway of protein secretion, in which newly synthesized protein is processed by the Golgi, packaged into small vesicles, and immediately secreted without intracellular storage. In the case of lymphokine secretion by T lymphocytes, it is generally not clear whether this non-regulated pathway is also being used, as opposed to the "regulated" pathway which has been proposed to operate in the cytotoxic lymphocyte mechanism. In this case, as in mast cells and endocrine cells, proteins are synthesized and then stored in cytoplasmic granules. The secretion is triggered (regulated) by a membrane receptor-ligand interaction, which for the cytotoxic lymphocytes is part of the target cell binding process. In cytotoxic T lymphocytes, this secretion process can be measured by following the appearance of a granule serine protease in the medium, and it has been shown to be triggered by target cells or by immobilized antibodies which bind the T cell receptor complex. In addition to cytotoxic lymphocytes, cloned T helper cells contain this serine protease in cytoplasmic granules with a low internal pH. Helper lymphocytes secrete this enzyme in response to (1) soluble antigen which has been processed by cells bearing the appropriate MHC antigens; (2) immobilized antibodies against the T cell receptor complex; (3) a combination of phorbol ester and calcium ionophore. Thus in both helper and cytotoxic lymphocytes, the regulated pathway of protein secretion clearly operates after triggering by the T cell antigen receptor.

Animals↗
PubMed1986

NK-leukocyte chemotactic factor (NK-LCF): a large granular lymphocyte (LGL) granule-associated chemotactic factor.

A chemotactic factor was identified in the supernatants of human large granular lymphocytes (LGL) activated by a glutaraldehyde-fixed NK-sensitive tumor, K562. The factor stimulated migration of human LGL, rat alveolar macrophage (RAM), and human monocytes and neutrophils (PMN). The locomotor response was chemotactic and chemokinetic on the basis of unidirectional migration in concentration gradients. The cell producing the factor was detected exclusively in LGL-rich Percoll fraction coincident with the peak of NK lytic activity and HNK-1+ cells. The monoclonal phenotype of the cell was HNK-1+, partially OKT-11+, OKM-1-, OKT-3-, OKT-4-, and OKT-8-. The factor was released by LGL within 20 min of incubation with Sr++, a cation that is able to induce LGL degranulation. A powerful chemoattractant was also detected in the granules of the rat LGL leukemia, RNK. Chemotactic activity coincided with granule enzyme beta-glucuronidase and cytolysin after RNK nitrogen cavitation and Percoll fractionation of subcellular constituents. The RNK granule chemoattractant induced unidirectional migration of human LGL and was also active against rat alveolar macrophages and human PMN. Anti-RNK granule antibody conjugated to Sepharose 4B was able to deplete the chemotactic activity from both K562-induced LGL supernatants and solubilized RNK granules. These observations indicate that a leukocyte chemotactic factor (NK-LCF) is present in NK cell granules and is probably released after tumor-induced granule exocytosis.

Chemotactic Factors↗
PubMed1983

Enhancement of the recognition by cytotoxic T lymphocytes (CTL) of target membrane antigens after fusion with whole cells.

Plasma membrane vesicles (R4-PM) prepared from mouse lymphoma cells (RDM4,H2k) were employed to investigate requirements for recognition of target cell membranes by allogeneic cytotoxic T lymphocytes (CTL). Using immunofluorescent staining and fluorescence microscopy, the R4-PM were tested for binding to CTL and were found to bind to these effector cells in a specific manner. However, this binding was very inefficient compared to the binding of whole RDM4 cells to CTL. The R4-PM were then attached to P388D1 cells (H-2d) in the presence of wheat germ agglutinin and polyethylene glycol (PEG), both under conditions which promote membrane fusion (40% PEG) and under conditions which do not (10% PEG). About 1 cell equivalent R4-PM becomes associated per P388D1 cell in both situations. In the cytotoxicity assays that were carried out, the P388D1 cells which had R4-PM attached under fusion conditions were lysed by CTL directed against H2k in a specific manner, while the P388D1 cells which had R4-PM attached under nonfusion conditions were not lysed above background levels by these CTL. These results suggest that recognition of target cells by allogeneic CTL such that lysis occurs requires more than presentation of the alloantigens as they are expressed in plasma membrane vesicles. However, fusion of these vesicles back into living cells apparently enhances the ability of the alloantigens to be recognized.

Animals↗
PubMed1982

Langerhans cell-containing epidermal cells are not cytotoxic in antibody-dependent- and mitogen-induced-cellular cytotoxicity.

Langerhans cells (LC) constitute a small (2-6%) subpopulation of mammalian epidermal cells (EC) and are the only EC that bear Fc-IgG receptors and that share several other cell-surface characteristics and functional capabilities with cells of the monocyte-macrophage series. Since Fc-IgG receptor-positive macrophages are potent effector cells in antibody-dependent cellular cytotoxicity (ADCC), we determined whether LC-containing EC populations are also able to mediate ADCC. In the ADCC assay employed, BALB/c LC-containing EC at different effector/target cell ratios consistently failed to lyse bovine red blood cell (BRBC) targets which were either trinitrophenyl(TNP)-modified or not modified and reacted with an appropriate antibody: a purified rabbit IgG anti-BRBC, or a mouse or rabbit anti-TNP antiserum. In addition, LC-containing EC did not mediate mitogen-induced cellular cytotoxicity using purified phytohemagglutinin. BALB/c spleen cells, used as a positive control, gave substantial specific release in all experiments performed.

Animals↗
PubMed1982

Induction of cell-mediated cytotoxic self responses by epidermal cells modified with a haptenic sulphydryl reagent.

Murine epidermal cells (EC) act as stimulator cells in the generation of allogeneic cytotoxic T lymphocytes (CTL) in cell-mediated lympholysis (CML) and are suitable targets for allogeneic and hapten-self CTL. To analyse the role of EC in the generation of and recognition by anti-self CTL, syngeneic hapten-modified murine EC were used as in vivo and in vitro stimulating populations and as target cells in a hapten-self CML system. Epidermal cells were modified with the sulphydryl-reactive haptenic reagent N-iodoacetyl-N'-(5-sulphonic-1-naphthyl)ethylenediamine (I-AED). C3H.SW (H-2b) AED-self CTL responses were generated by stimulation with syngeneic AED-modified EC and were readily demonstrated when tested on syngeneic hapten-modified EC. These CML responses were hapten-specific and H-2 restricted. No substantial difference was detected in the ability of AED-modified EC and spleen cells (SC) to stimulate the generation of secondary AED-self CTL. Cold target inhibition experiments with hapten-modified EC and SC blockers did not reveal tissue-specific recognition of hapten-modified EC or SC targets by AED-self CTL. These findings demonstrate that hapten-modified EC, when used for priming in vivo and subsequently for in vitro sensitization, can induce hapten-specific self CTL that are reactive against syngeneic hapten-modified EC.

Animals↗
PubMed1981

Spatially distinct allodeterminants of the H-2Kk molecule as detected by monoclonal anti-H-2 antibodies.

In an attempt to delineate spatial relationships between various allodeterminants of cell surface MHC antigens, competitive binding studies were performed using 4 different monoclonal antibodies, each of which reacted with the H-2Kk antigen but with different serologic specificities. Competition was studied by examining the effect of unlabeled antibodies on the binding of each 125I-labeled antibody to spleen cells of the H-2a haplotype. Mutual inhibition was observed between 2 of the antibodies, and a 3rd antibody of lower affinity was inhibited by the first 2 antibodies but did not itself inhibit the binding of these antibodies. The 4th antibody did not block the binding of the other 3 labeled antibodies, and binding of this 4th labeled antibody was only partially inhibitable by the other 3 antibodies. These results indicate the presence of at least 2 spatially distinct allodeterminants on H-2Kk molecules expressed on the cell surface.

Animals↗
PubMed1980

Diffusion, patching, and capping of stearoylated dextrans on 3T3 cell plasma membranes.

Fluorescence-labeled trinitrophenylated stearoylated dextrans have been used as controllable analogues of cell membrane proteins on model membranes and on a variety of natural cell membranes. This paper reports their behavior on 3T3 mouse fibroblast plasma membranes. Spatial distribution on the membrane was studied by fluorescence microscopy, and molecular mobility was measured by fluorescence photobleaching recovery. At concentrations from 10(2) to 3 X 10(3) molecules/micron2 essentially homogeneous fluorescence was observed after treatment with these stearoyldextrans in culture. Diffusion coefficients and fractional recovery of fluorescence after photobleaching were cvoncentration independent. For 3 X 10(3) molecules/micron2 we found at 23 degrees C D = (3.0 +/- 1.8) X 10(-10) cm2/s with 65 +/- 17% recovery and at 37 degrees C D = (7.0 +/- 5.0) X 10(-10) cm2/s without a change of the fractional recovery. Cross-linking with antibodies stopped diffusion on a macroscopic scale and sometimes induced patching, mottling (defined as the development of gaps in the fluorescence layer), and capping (defined as the confinement of the fluorescence to less than 50% of the cell). Capping required approximately 3 h at 37 degrees C and was inhibited by metabolic poisons and cytochalasin B. These drugs did not affect stearoyldextran diffusion or fractional recovery. Colchicine, which did not dramatically affect capping, slowed diffusion two- to threefold but did not affect fractional recovery. The antibody inhibition of the diffusion of stearoyldextrans precedent to capping did not affect the diffusion of a lipid probe or fluorescein isothiocyanate labeled membrane proteins. When the trinitrophenylated stearoyldextran was cleared from most of the surface by capping and the surface subsequently relabeled with stearoyldextran, the diffusion coefficient and fractional recovery of the second label were identical with those of the first label prior to capping. Thus, capping does not clear an immobilizing factor from the membrane.

Animals↗
PubMed1980

Lysophosphatidylcholine in liposomal membranes: enhanced permeability but little effect on transfer of a water-soluble fluorescent marker into human lymphocytes.

In an attempt to enhance delivery of liposome contents into cells, we tested the effect of lysophosphatidylcholine on transfer of the fluorescent dye, carboxyfluorescein, from small unilamellar and large multilamellar vesicles to human lymphocytes. Dioleoyl phosphatidylcholine and dioleoyl phosphatidylcholine-lysophosphatidylcholine small unilamellar vesicles with varying lipid ratios were prepared and characterized. In the presence of lysophosphatidylcholine, small unilamellar vesicles were slightly smaller and more leaky than those made without lysophosphatidylcholine. Lysophosphatidylcholine induced less leakage in large multilamellar vesicles. It did not show any appreciable effect on transfer of liposome contents, whether included as part of the liposomal bilayer (of unilamellar or multilamellar vesicles) or added exogenously together with small unilamellar dioleoyl phosphatidylcholine vesicles.

Cell Membrane Permeability↗
PubMed1980

Electron microscopic demonstration of lesions in target cell membranes associated with antibody-dependent cellular cytotoxicity.

To test the hypothesis that complement-mediated cell lysis and cell-mediated cytotoxicity operate by analogous mechanisms, cell membranes from two antibody-dependent cytotoxicity systems were examined by electron microscopy after negative staining. Ring-shaped membrane lesions generally similar to, but larger than, those previously described for complement lysis were observed. These findings are in agreement with recent measurements of larger functional pores for ADCC than complement.

Antibody-Dependent Cell Cytotoxicity↗