The biochemistry of lymphocyte-derived mediators of immunological inflammation

Evidence is presented to indicate that there exists in lymphoid tissue, as a result of transforming lymphocytes, a new lymphokine which is chemotactically specific for lymphocytes, called lymphotactin. Lymphotactin has been purified to electrophoretic homogeneity; has a molecular weight of 10,500 D and an isoelectric point of 5.9. Its role in amplifying the immune defense system by recruitment of naive lymphocytes into propinquity with the challenging antigen is suggested.Purification of macrophage migration inhibitory factor from thymus extracts to electrophoretic homogeneity leads to a compound of molecular weight of 36,500 D and an IEP of 6.9. Chemically it contains sialic acid ando-methyl glucopyranoside as its only carbohydrates. Purified MIF activates the macrophage phagocytically.Skin reactive factor and lymph node permeability factor have been isolated and purified and are found to be inhibited by pepstatin and antihistamine and to have an isoelectric point of pH 4.2 and a molecular weight of 50,000–100,000 D. It is believed that this anionic permeability increasing agent actually arises from the lysosomes of macrophages and lymphoblasts (the normal small lymphocyte having essentially no lysosomal organelles). The mononuclear cell infiltration characteristic of crude SRF and LNPF may proceed from their being contaminated with lymphotactin.     

Cathepsin D-like activity in neutrophils and monocytes.

Monocytes-macrophages and polymorphonuclear leukocytes contain an acid proteolytic enzyme that cleaves tritiated hemoglobin. The monocyte-macrophage-derived enzymatic activity was completely inhibited by pepstatin A, a property of cathepsin D. Monocyte-derived macrophages developed detectable cathepsin D-like activity after 5 days in culture, and this activity coincided with the appearance of other known indicators of macrophage maturation. The cathepsin D activity further increased significantly with time after day 5 of culture. The proteinase activity extracted from neutrophils was only partially inhibitable by pepstatin A, which indicates that this activity is contributed by more than one proteolytic enzyme, including cathepsin D. Cathepsin D activity demonstrated in neutrophils and macrophages may be an important marker of phagocyte function.     

Permeability factor contaminating hyaluronidase preparations

Both crude and highly purified testicular hyaluronidase preparations have been shown to contain a component which increases the permeability of the microcirculation in rat skin. This permeability activity had an isoelectric point of 7.4 while hyaluronidase was 9.4. It also could be separated from hyaluronidase by acrylamide gel electrophoresis. The permeability factor was not inhibited by serum and may explain previous observations that hyaluronidase preparations decrease the ischemia and necrosis appropriate to experimental myocardial infarction in vivo.     

A proteaselike permeability factor in guinea pig skin: contact activation of the latent permeability factor and its nature as a prekallikrein activator.

A proteaselike permeability factor in guinea pig skin, which was fractionated in a latent form into pseudoglobulin fractions, was activated by contact with kaolin particles at neutral pH. This contact activation was not prevented by the presence of 1 M KCl but was strongly inhibited by the simultaneous presence of hexadimethrine bromide. In this activation, the latent permeability factor was first bound to kaolin; later an active form permeability factor was released from the kaolin-bound parent molecule. Prekallikrein activator activity was also generated in this supernatant from the pretreated kaolin particle in the same time course as the permeability factor generation. Moreover, since the prekallikrein activator and permeability factor were always observed at the same fractions in every purification step, with DEAE-cellulose column chromatography, Sephadex G-75 gel filtration, and isoelectric focusing, these two molecules were recognized as identical. These results indicate that the latent permeability factor in the guinea pig skin has properties similar to those of the plasma Hageman factor.     

A Factor Capable of Increasing Vascular Permeability Present in Lymph Node Cells: A Possible Mediator of the Delayed Reaction

A soluble extract from guinea-pig lymph node cells (LPF) has been found to increase vascular permeability in the skin of the rat. The active substance has been differentiated from histamine, 5-hydroxytryptamine, bradykinin, substance P, kallikrein and the globulin permeability factors from rat and guinea-pig serum by means of parallel quantitative assays. LPF was present in both sensitized and non-sensitized guinea-pig lymph node cells and in lymph node cells from rats and mice. LPF also increased vascular permeability in the skin of guinea-pigs, mice and rabbits. The possible importance of this factor in the mechanism of the delayed reactions is discussed.     

Macromolecular,anionic pulmonary permeability factor

Aqueous extracts of fresh or acetone-powdered calf lung have been found to contain a factor which increases the permeability of the microcirculation when injected into the skin of rats. This permeability factor, which is not found in similar extracts of muscle or kidney, was concentrated by Amicon ultrafiltration at a molecular weight range of between 50,000 and 100,000 daltons and via isoelectric focusing at an IEP of pH 4.2. After preparative acrylamide gel electrophoresis, this permeability factor was homogeneous by electrophoretic criteria and in SDS acrylamide gel electrophoresis had a molecular weight of approximately 82,000 daltons. This apparently homogeneous permeability factor from lung was inhibited by pepstatin, and yet possessed no acid proteolytic activity against any substrate. Its activity was completely inhibited by pretreatment of the animals with antihistamines. This pepstatin-inhibitable permeability factor was found largely in the lysosomal fraction of fresh lung and could also be obtained by extraction of large amounts of alveolar washout macrophages. Since macrophages, during phagocytosis, are known to leak significant quantities of their lysosomal contents, the fact that one of these components is a material which could release histamine from mast cells and thereby increase locally the permeability of the microcirculation may be of importance in the defense system of the lung.Supported in part by NIH BRSG grant # RP05588-11, ONR N00014-77 C02062, the Washington Lung Association, and grant HL 22369.     

Lymph node permeability factor in the dinitrochlorobenzene skin hypersensitivity reaction in guinea-pigs

The vascular permeability factor present in extracts of lymph node cells (LNPF) has been examined as a possible mediator of the cutaneous inflammation associated with the delayed hypersensitivity produced by painting the skin of guinea-pigs with 2:4-dinitrochlorobenzene.

It has been found that during the development of the DNCB reaction it becomes possible to extract from the reaction site a substance indistinguishable from LNPF, the results being similar to those obtained previously in tuberculin sensitivity. Precipitating antibody to guinea-pig LNPF has been produced in rabbits. Pretreatment of animals with the antibody serum caused a marked diminution of the erythema and cellular infiltration seen after application of the challenging dose of DNCB.

It is suggested that in the tuberculin and DNCB reactions, sensitized mononuclear cells may reach the reaction site as a result of non-specific stimuli, react with antigen and release LNPF which might bring about a major inflammatory response.

     

Eosinophil chemotaxis of supernatants from cultured Hodgkin's lymph node cells.

Cultured lymph node cell supernatants from five out of six cases of Hodgkin's disease were relatively more chemotactic for peripheral blood eosinophils than neutrophils. In contrast, supernatants from two cases of lymphocytic lymphoma and four nodes showing reactive hyperplasia were chemotactic for neutrophils but had little eosinophil chemotactic activity. In most instances the dgree of eosinophil infiltration observed histologically in the Hodgkin's lymph node correlated with elaboration of eosinophil chemotactic activity from the cultured cells. Following gel-filtration of three of the Hodgkin's lymphnode supernatants, four peaks of eosinophil chemotactic activity were demonstrated in each case. One of these corresponded in molecular size to th previously described eosinophil chemotactic factor of anaphylaxis (ECF-A). It is suggested that the eosinophil chemotactic activity of cultured lymph node cell supernatants may be on value in the diagnosis and classification of Hodgkin's disease.     

Purification of Plasmodium lophurae cathepsin D and its effects on erythrocyte membrane proteins

The cathepsin D of Plasmodium lophurae was purified using a combination of CM-Sephadex, pepstatin-agarose and Sephadex G-100 chromatography. The plasmodial enzyme was distinct from that of the host red cell and bovine spleen in its low isoelectric point (pI 4.3). The cathepsin D of P. lophurae, as well as plasmodial extracts demonstrating such proteinase activity, were able to digest the membrane proteins of duckling and human red cells at pH 7.4; proteolysis was not inhibited in phosphate-buffered saline by 100 microM pepstatin. Membrane proteins most susceptible to proteolysis were those of the cytoskeleton, notably bands 1 and 2 (spectrin), bands 2.1-2.6 (spectrin-binding proteins) and band 3. Membrane protein degradation by crude plasmodial extracts was partially inhibited by a combination of 10 mM FeCl3, and 10 mM phenylmethylsulfonyl fluoride in phosphate-buffered saline. The changes induced in erythrocyte membrane proteins by exposure to plasmodial cathepsin D parallel the alterations observed in red cell membranes obtained from malaria infected cells. Since the action of the plasmodial protease was confined to the inner surface of the red cell membrane, it is possible that protease-induced modifications in the red cell cytoskeleton could lead to merozoite release.     

Purification of serum proteins from lymph node cell extracts with permeability activity characteristic of the lymph node permeability factor

The biologically active aqueous extract of rat lymph node cells, previously shown to contain the lymph node permeability factor LNPF, was fractionated by gel filtration and ion exchange chromatography. The fractions obtained were characterized by immunoelectrophoretic analysis and found to contain a wide range of serum proteins. The ability of the fractions to increase vascular permeability to plasma protein was measured. Activity was found to be distributed among several serum proteins and was mainly associated with albumin, though minor quantities of γ-globulin were also found to have high specific activity. Comparative analysis of γ-globulin and albumin prepared from lymph node cell extract and serum, respectively, suggested that the active factor was most likely to be released from the lymph node cells to form complexes with these proteins.     

Regulatory dysfunction in leukotaxis.

The chemotactic factor inactivator (CFI) in human serum appears to have important regulatory function in the inflammatory response. In humans with elevated serum levels of CFI, defective mobilization of leukocytes in vivo has been noted, both in skin windows and in skin testing with various antigens. In experimental immune complex-induced acute inflammatory reactions in rat skin and lung, purified human CFI at very low doses has potent antiinflammatory effects and is able to suppress permeability changes, neutrophil infiltration, and hemorrhage, all of which are dependent initially on the role of leukotactic mediators.     

A protease-like permeability factor in guinea pig skin: immunologic identity with plasma Hageman factor.

Vascular permeability enhancement activity of the protease-like permeability factor derived from guinea pig skin and of active guinea pig Hageman factor (beta HFa) were both inhibited by anti-guinea pig Hageman factor rabbit F(ab')2 antibody. The permeability activity of both factors was also absorbed on anti-Hageman factor F(ab')2-Sepharose beads. The latent form of the permeability factor derived from skin extracts produced a single immunoprecipitation line with anti-Hageman factor and gave a reaction of identity with a precipitation band developing between purified Hageman factor and anti-Hageman factor. The latent permeability factor in the fraction corrected the clotting activity of Hageman-factor-deficient human plasma. The clotting activity was also blocked by anti-Hageman factor F(ab')2 antibody. From these results, it was concluded that the skin permeability factor was immunologically and functionally indistinguishable from Hageman factor of plasma. Extracts were obtained from skin of guinea pigs given intravenous injections of 125I-guinea pig Hageman factor immediately before sacrifice to calculate the amount of Hageman factor in the extravascular tissue space of the skin. The pseudoglobulin fractions of the extracts containing a concentration of Hageman factor of approximately 9 microgram of Hageman factor per gram of skin. This was determined both by immunologic means and procoagulant activity. Only 4% of the Hageman factor in the extract was obtained from the intravascular plasma volume of the skin.     

Regulation of eosinophilia in rats infected with Nippostrongylus brasiliensis. I. Eosinophil chemotactic factor produced spontaneously by mesenteric lymph node cells of infected rats

The possible mechanism of eosinophilia was studied in rats undergoing primary infection with Nippostrongylus brasiliensis (Nb). In vivo studies showed that the kinetics of intestinal tissue eosinophilia was not directly related to those of the intestinal worm burden. Furthermore, Nb worm extract has no or only very weak in vitro eosinophil chemotactic activity, suggesting that parasite-derived eosinophil chemotactic factor (ECF) is, if at all, not a major regulator for intestinal tissue eosinophilia in this Nb rat system. On the other hand, when mesenteric lymph node (MLN) cells obtained various days after infection were cultured, potent ECF activity was detected in the cell-free supernatant from the cultures of MLN cells 15-20 days after infection, at which time marked intestinal tissue eosinophilia was observed in vivo. Production of ECF by MLN cells from Nb-infected rats seems to be spontaneous, since these cultures were performed without adding worm antigen. ECF-producing activity of day-20 MLN cells was suppressed by adding various metabolic inhibitors such as cycloheximide, mitomycin C, or puromycin. After Sephadex G-75 gel filtration, ECF activity produced by day-20 MLN cells was associated with two different molecules.     

Bactericidal activity of rabbit serum containing immunoconglutinin

When lymphocytes from hypersensitive animals are incubated with antigen, biologically active substances are formed which inhibit the migration of mesenchymal cells from normal animals.

These substances were tested by intradermal injection in guinea-pigs and rabbits. The supernatants from incubation of lymphocytes with a high dose of antigen caused immediate pallor which lasted several hours. Later there was a macroscopic inflammation maximal at 24 hours. The histology was characteristic of a delayed hypersensitivity reaction.

The injection of the supernatant from hypersensitive lymphocytes incubated with a small dose of antigen caused little or no pallor and was not followed by a delayed inflammatory reaction. Injection of this supernatant together with the antigen did not potentiate or alter the reaction, in contrast to in vitro experiments where the inhibition of the migration by this supernatant was potentiated by antigen.

Besides this factor a distinct factor occurs in extracts and supernatant fluids of lymphocytes cultivated without antigen and those from control animals, which increases vascular permeability. This substance is probably identical with the lymph node permeability factor (LNPF). The possible role of these biologically active substances in the mechanism of delayed type hypersensitivity is discussed.

     

Pulmonary polyamine permeability factor

Acid extracts of calf lung have been found to contain low-molecular-weight factors which increase the permeability of the microcirculation when injected into the skin of rats. These factors, which were present in very low levels in aqueous extracts, were purified by gel filtration and ion-exchange chromatography. High-voltage paper electrophoresis revealed two active compounds with mobilities identical to the polyamines spermine and spermidine. Authentic samples of these compounds were as active in the blueing reaction as the isolated compounds. The permeability activity of both the isolated factors and the synthetic ones was inhibited by pepstatin and by pretreatment of the animals with pyrilamine maleate. If the normally low extracellular levels of these polyamines is increased by tissue damage, they could increase vascular permeability within the lung by releasing histamine from adjacent mast cells.     

组织蛋白酶D新的糖基化异构体在肺癌中表达的临床意义及其与预后的相关性

目的 探讨组织蛋白酶D（CTSD）新的糖基化异构体与肺癌临床病理特征及预后的相关性。 方法 应用免疫组织化学和半定量RT PCR方法检测119例肺癌组织和39例癌旁组织中CTSD的表达;Western blotting分析CTSD各异构体表达特点;去糖基化实验检测CTSD 蛋白的糖基化修饰;Kaplan-Meier分析66kD异构体、临床病理资料与肺癌术后整体生存期的相关性。 结果 CTSD在肺鳞癌、腺癌和小细胞肺癌组织中高表达;66kD蛋白为CTSD新的糖基化异构体,该异构体表达与肺癌组织类型、临床分期、淋巴结转移和患者的吸烟史密切相关（P<0.05）,阳性表达和阴性表达患者术后中位生存时间分别为20.0和30.0个月（P<0.05）;肺癌组织类型、临床分期、淋巴结转移、肿瘤大小和66kD异构体表达是影响肺癌预后的独立因素。 结论 CTSD 66kD异构体阳性表达可能是肺癌预后不良的分子标志物之一。     

Immunohistochemical detection of nm23/NDP kinase and cathepsin D in medullary carcinomas of the thyroid gland

Reduced expression of nm23/NDP kinase and increased expression of cathepsin D seem to be correlated with the high metastatic potential in a variety of malignancies. The expression of nm23/NDP kinase and that of cathepsin D have been evaluated by means of an immunohistochemical technique in paraffin-embedded tissues from 44 primary medullary carcinomas of the thyroid gland (MCT) and from the corresponding lymph node metastases in 32 of these cases. In addition, lymph node metastases from 4 cases were studied. We found that 36 of 44 (82%) primary and 26 of 36 (72%) lymph node metastatic MCT were nm23/NDP kinase positive, whereas 14 of the 44 (32%) primary and 17 of the 36 (47%) lymph node metastatic MCT were cathepsin D positive. We found no indication that the nm23/NDP kinase level has any prognostic significance in MCT. The cathepsin D level is close to being prognostically significant in this study, and we cannot exclude the possibility that it could be of prognostic value. However, it seems to be quite weak, and therefore of little use in a clinical situation.     

The chemotactic properties of cartilage glycosaminoglycans for polymorphonuclear neutrophils

Cartilage breakdown products have been tested for their effects on the locomotion of rat polymorphonuclear neutrophils in vitro. Chondroitin sulphate, keratan sulphate and hyaluronic acid were used in checkerboard migration assays to differentiate chemotaxis from chemokinesis. All test substances showed both chemokinetic and chemotactic activities. The same substances were then injected intradermally to determine their effects on vascular permeability and leukocyte accumulation in vivo. There was no significant effect on vascular permeability 30 minutes after injection as measured by a dye-leakage method. Histological examination of skin sections taken 6 hours after injection showed modest accumulations of polymorphonuclear neutrophils. It is suggested that cartilage breakdown products may account for the persistence of polymorphonuclear neutrophils in some chronic inflammatory joint diseases.     

Studies on guinea-pig macrophage migration inhibitory factor (MIF). I. Glycoprotein nature and net charge.

Guinea-pig macrophage migration inhibitory factor (MIF), obtained by the stimulation of sensitized lymph node cells with tuberculin PPD, was characterized as a glycoprotein by the following criteria: (a) its activity is destroyed by 0.02 M sodium periodate; (b) when MIF-containing culture fluids are subjected to precipitation by perchloric acid (final concentration 1 M), the inhibitory activity is recovered in the supernatant; and (c) MIF binds to Sepharose-linked concanavalin A and can be eluted with methyl-alpha-D-glucopyranoside. When MIF-containing culture supernatants are fractionated by isoelectrofocussing, migration inhibitory activity is recovered in a fraction with an isoelectric point of 4.4--4.6.     

小鼠SLC基因真核表达载体的构建及其趋化活性鉴定

目的：克隆小鼠次级淋巴样组织趋化因子(secondary lymphoid-tissue chemokine，SLC)基因，并构建真核表达载体。在体内外检测该表达载体表达产物的免疫趋化功能。方法：用RT—PCR从C57BL／6小鼠胸腺组织中克隆小鼠SLC基因，构建真核表达载体pcDNA3．1—msLC。在体外，用基因枪转染小鼠黑色素瘤B16F10细胞；RT—PCR检测转染细胞有SLC的表达；利用趋化小室法，检测表达产物针对淋巴细胞的趋化活性。在体内，用基因枪通过小鼠皮肤局部转染SLC基因，观察转染局部淋巴细胞的浸润。结果：克隆的基因经测序证实，为小鼠SLC基因Scya21b型。转染SLC基因的B16F10细胞体外培养48h后，RT—PCR检测到SLC的表达，同时培养基上清具有针对淋巴细胞的趋化活性。通过基因枪局部转染小鼠皮肤，24h后皮肤病理检查显示，局部皮内有明显的淋巴细胞浸润。结论：从小鼠胸腺组织中克隆到小鼠SLC基因，构建的真核表达载体pcDNA3．1—mSLC在体内外均可以表达，并具有针对淋巴细胞的趋化活性。