Zanvil A. Cohn

Purification and characterization of a cytolytic pore-forming protein from granules of cloned lymphocytes with natural killer activity

A cytolytic pore-forming protein (PFP, perforin) was purified from isolated granules of cloned NK-like cytolytic cells, which showed an apparent Mr of 70-75 kd (reduced) and 62-66 kd (nonreduced). Cytolysis produced by this protein occurred only in the presence of Ca2+ and was accompanied by the formation of membrane lesions of 160 A diameter. The purified protein depolarized cells and made lipid vesicles leaky to monovalent and divalent ions. This protein formed large, voltage insensitive and nonselective ion channels in planar bilayers that remained preferentially in the open state. The channels were heterogeneous in size distribution averaging 400 pS/U in 0.1 M NaCl. The membrane lesions formed by PFP were morphologically and functionally similar to those formed by intact NK-like cells and their granules. This PFP could be released from granules during cell killing, followed by its polymerization on target membranes to form large transmembrane pores.

Local and Systemic Effects of Intradermal Recombinant Interferon-γ in Patients with Lepromatous Leprosy

Evidence that interferon-gamma may be a physiologic macrophage-activating factor, and that macrophage activation may be defective in lepromatous leprosy, led us to test the effects of intradermal injection of low doses of recombinant interferon-gamma in six patients with this disease. Interferon-gamma, 1 or 10 micrograms, was administered daily by jet gun for three days into a single cutaneous lesion. A biopsy specimen was taken from the injection site on the sixth study day and compared with specimens obtained previously from a site where no injection had been made or where excipient alone had been injected in the same way as the interferon. Interferon-gamma elicited local effects similar to certain features of delayed-type hypersensitivity reactions or tuberculoid leprosy, including induration, T-cell and monocyte infiltration, keratinocyte proliferation, diminution of epidermal Langerhans cells, and dermal and epidermal cell HLA-DR (Ia) antigen expression. At some of the sites of interferon-gamma injection, there was also an apparent decrease in acid-fast bacilli. Before treatment, monocytes from patients with lepromatous leprosy released 48 percent as much hydrogen peroxide as did monocytes from controls in response to phorbol myristate acetate, and 36 percent as much as those from controls in response to Mycobacterium leprae. When recombinant interferon-gamma was injected, these responses became normal. No toxic effects were observed. These observations suggest that interferon-gamma can mediate certain manifestations of delayed-type hypersensitivity or cell-mediated immunity in vivo, and that recombinant interferon-gamma should be tested for possible therapeutic effects in certain nonviral infectious diseases.

The structure and function of monocytes and macrophages.

Publisher Summary This chapter discusses the more recent advances in the biology and chemistry of the blood monocytes and tissue macrophages and mechanisms of immunological importance. The mononuclear phagocytes that comprise the reticuloendothelial system represent a large, widely distributed and morphologically heterogeneous group of cells. Originating from the mesoderm during embryogenesis they form a part of all tissues and are particularly prominent in the bone marrow, liver, spleen, connective tissues, serous cavities, and blood. They are involved in diverse functions, related in large measure to their endocytic activities and to the intracytoplasmic digestion that is a consequence of their heterophagic function. Much of the work on the metabolism of the monocytes and macrophages represents ancillary information obtained through the study of the functional attributes of the cell. Relatively few investigators have examined the properties of the blood monocyte because of the difficulties of obtaining large enough samples. The more mature macrophage derives energy from both glycolytic and respiratory pathways. Aerobic glycolysis is active and a Pasteur Effect is seen in exudate macrophages.

Isolation and characterization of a serine esterase from cytolytic T cell granules

Cytotoxic T lymphocytes and lymphocytes with NK-like activity contain a serine esterase activity which has been localized to their cytoplasmic granules by cytochemistry and subcellular fractionation studies. The serine esterase-specific inhibitor 3H-DFP labels two protein species in the granules. The two proteins, referred to as serine esterases 1 and 2 (SE 1 and SE 2), migrate with Mr of 34-36 kd and 28-30 kd, respectively, under reducing conditions. SE 1 shows trypsin-like activity and has been purified to apparent homogeneity. Under nonreducing conditions, SE 1 has an Mr of 60-66 kd, suggesting that it may consist of two disulfide-linked subunits of 34-36 kd each. SE 1 cleaves fibrin and casein, has a pl greater than 10, and optimal activity at pH 8. The substrate specificity of SE 2 is not known. The serine esterase activity is secreted by lymphocytes that have been stimulated with the calcium ionophore A23187. The serine esterases described here could play an active role in cell-mediated killing.

Functional assembly of gap junction conductance in lipid bilayers: demonstration that the major 27 kd protein forms the junctional channel.

Gap junctions isolated from rat liver were incorporated into planar lipid bilayers. A channel activity that was directly dependent on voltage was recorded. Changes of pH and (Ca2+) had no direct effect on channel activity; however, they modulated the voltage-dependent gating of the gap junction channels differently. Single-channel fluctuations showed large scatter with peak amplitudes of 140 and 280 picoSiemmens in 0.1 M NaCl. The major protein of gap junctions (Mr of 27 kd) was also reconstituted into bilayers, giving channel properties similar to those of intact gap junctions. Polyclonal antibodies specific for this protein caused inhibition of the junctional conductance in bilayers. These data provide direct evidence that the 27 kd protein is the molecular species responsible for gap junction communication between cells.

Cellular and Humoral Mechanisms of Cytotoxicity: Structural and Functional Analogies

Publisher Summary Target cell (TC) killing, however, is not restricted to immune cells and has been documented for various cell types, including bacteria, fungi, yeast, plant cells, and protozoan parasites. Perhaps one denominator common to all these different cytotoxic cell types and their killing machineries is the involvement of soluble cytotoxic mediators that are secreted by the killer cell and used to lyse the target. The increased interest in these soluble mediators of cell killing, generally referred to as “cytotoxins,” has been greatly stimulated in part by the feasibility of their isolation in the laboratory in high yields and their further characterization by conventional biochemical techniques. This chapter focuses on the molecular mechanisms of membrane. The surprising functional analogies between the cytotoxin released by lymphocytes and the better known toxins found in certain bacteria and insects, for example, should greatly stimulate and aid future studies on the cytotoxic reaction mediated by immune cells.

Perforin‐Dependent and ‐Independent Pathways of Cytotoxicity Mediated by Lymphocytes

There is little doubt at the present time that both perforin-dependent and -independent pathways are important in mediating the cytotoxicity associated with lymphocytes. The cell distribution of perforin, initially thought to include both CTL and NK cells, now must be viewed with caution because all previous biochemical studies on CTL have been conducted with cell lines propagated in long-term cultures in the presence of T cell growth factors (IL-2 and perhaps some still undefined factors). Under these conditions, CTL are known to assume a broader, NK-like specificity in target cell killing and may thus differ significantly from primary CTL generated in the body. Accordingly, perforin does not seem to be present in primary CTL activated directly through mixed lymphocyte reactions. It remains to be shown how primary CTL lyse target cells in vivo. Initial studies conducted in several laboratories have already provided some clues. It now seems that even in cultured, perforin-containing CTL, the perforin pathway is not an obligatory mechanism required for target cell killing. Other pathways, possibly involving TNF/lymphotoxin-like molecules, may play a direct role in this type of cytotoxicity. Other still unidentified factors now also need to be sought, including membrane polypeptides that may develop cytotoxicity directly upon cell contact and binding. Although from the studies reviewed here it is clear now that perforin has a more limited role in cell killing than originally proposed, it is still intriguing that it should share structural and functional homologies with complement proteins, drawing paradoxical analogies between two systems (the cellular and the humoral immune systems) which have evolved to become specialized to carry out separate immunological tasks. The cloning of the genes for perforin and for all the C proteins that comprise the MAC should reveal important information on how these genes originated and then diverged during evolution. The cellular distribution of other granule products, such as serine esterases, also must be viewed with caution. A serine esterase activity was initially thought to be CTL-specific. This information stimulated an intensive research activity in many laboratories that resulted in both the purification of a serine esterase family and the cloning of several serine esterase transcripts. It is becoming clear from recent evidence that this group of enzymes is not truly CTL-specific and therefore would not be expected to develop any function rendered absolutely necessary for cytolysis.(ABSTRACT TRUNCATED AT 400 WORDS)

The treatment of sarcoidosis with chloroquine

Abstract Chloroquine has been administered to seven patients with chronic sarcoidosis. In each instance there was considerable improvement of the cutaneous lesions. Regression of extracutaneous lesions was more variable, but improvement was often observed; this was particularly true of mucous membrane and thoracic node involvement. Associated with the clinical response, elevated sedimentation rates and high gamma globulin levels returned toward normal. It is suggested that chloroquine may be a useful drug for the treatment of many patients with sarcoidosis, and further studies are in progress to define the situations in which its use is of most benefit.

Interaction of enveloped viruses with planar bilayer membranes: Observations on Sendai, influenza, vesicular stomatitis, and Semliki forest viruses

Exposure of a planar lipid bilayer to Sendai virus at pH 7.0 resulted in conductance increases that continued over several minutes, provided that the virus particles had first been conditioned by freezing and thawing, sonicating, or storing for 2 weeks in the cold. Individual electrical events could not be resolved, even on a millisecond time scale, and thus do not reflect the insertion of structural channels into the lipid bilayer. Prior treatment of the Sendai virions with protease prevented the conductance increases, but exposure of the bilayer to protease after induction of the conductance change did not abolish it. The Sendai-induced conductance change was increased in rate, but qualitatively unchanged in nature, if gangliosides were included in the planar bilayer. Activity for Sendai virus was low at pH 5.0, and increased with increasing pH up to 9.0. Influenza, Semliki Forest virus, and vesicular stomatitis virus all induced similar conductance changes around pH 5.2, but were inactive when tested at pH 7.0. The presence of cholesterol in the bilayer caused marked enhancement (two- to sixfold) of the response to Sendai, influenza and Semliki Forest virus, but caused only slight enhancement of the response to vesicular stomatitis virus. It is concluded that the observed increases in ionic permeability arise from alterations in lipid motions on a submillisecond time scale resulting from the incorporation of damaged viral membranes into the planar bilayer by fusion.

THE METABOLISM AND PHYSIOLOGY OF THE MONONUCLEAR PHAGOCYTES

Publisher Summary This chapter provides an overview of the metabolism and physiology of mononuclear phagocytes. The two general categories of mononuclear phagocytes are those that are found circulating in blood and those that are found in the fixed and wandering cells of tissues. The morphologic characteristics of the mononuclear phagocytes constitute important criteria for classification and evaluation of functional activity. There are many transitional forms between the classic blood monocyte and tissue macrophage. In a usual blood film, the monocyte is the largest mononuclear cell, with diameters ranging from 12 to 15 microns. The tissue macrophages differ markedly from monocytes and exhibit a much greater morphologic heterogeneity. One basic difference is their large size, which can vary from 15 to 80 microns. Following the parenteral administration of vital dyes, colloids, or large particles, a variety of cells can be demonstrated that contain these materials. These cells vary in morphology when observed in situ but have in common the ability to segregate dyes or phagocytize solids. They are found in almost all areas of the body but are usually concentrated in certain organs and are often in contact with vascular or lymphatic channels.