Cellular and humoral hypersensitivity reactions during silicosis and silicotuberculosis

Together with the synthesis of specific antibodies and autoantibodies, effects of cellular allergy, such as damage to neutrophils, agglomeration of leukocytes and inhibition of migration of leukocytes with tuberculin and isologous pulmonary antigen were established in patients with silicosis and silicotuberculosis. Autoallergy becomes manifest already in the "pre-roentgenological" stage of development of silicosis; in silicotuberculosis it correlates with the stage of the specific process. In both silicosis and silicotuberculosis, it includes elements of immediate and delayed hypersensitivity. In clinical practice, allergic reactions of leukocytes with tuberculin can be of assistance in determining the stage of the specific process and in differential diagnosis of silicosis and silicotuberculosis.     

Suppression of lymphocyte stimulation by anterior hypothalamic lesions in the guinea pig

Anterior hypothalamic lesions in the guinea pig inhibited lymphocyte stimulation in whole blood cultures with the antigen tuberculin and with the mitogen phytohemagglutinin (PHA) and suppressed the delayed cutaneous hypersensitivity response to tuberculin. The lesions did not affect the stimulation of purified lymphocytes with either tuberculin or PHA. The anterior hypothalamic lesions had no effect on the absolute number of T and B lymphocytes.     

Low-dose chymotrypsin treatment inhibits neutrophil migration into sites of inflammation in vivo: effects on Mac-1 and MEL-14 adhesion protein expression and function

Antibody blocking studies in the mouse suggest that the MEL-14 antigen is involved in neutrophil-endothelial cell interactions and may be important in neutrophil extravasation to sites of inflammation in vivo. We recently showed that chemotactic factor activation causes a rapid (within minutes) shedding of a large fragment of the MEL-14 antigen from the surface of neutrophils. We report here that chymotrypsin, at low doses (0.1 units/1 x 10(6) cells), but not trypsin, elastase, or collagenase, causes an activation-independent rapid loss (greater than 90%) of the MEL-14 antigen from the surface of murine neutrophils. Under the same treatment conditions chymotrypsin has no effect on the expression of four other neutrophil surface antigens, including the Mac-1 adhesion protein. Chymotrypsin treatment has no effect on neutrophil adhesion to plastic, migration to C5a, regulation of the Mac-1 antigen, but causes a greater than 95% reduction in neutrophil binding to high endothelial venules (HEV) in peripheral lymph nodes measured in the ex vivo frozen section HEV binding assay. The level of inhibition of neutrophil adhesion to HEV was comparable to that seen with the MEL-14 antibody. This experimental system allows us for the first time to specifically examine the consequences of removing the MEL-14 antigen from the surface of neutrophils on function in vivo. We show that treatment with chymotrypsin blocks greater than 85% of the ability of neutrophils injected back into the animal to home to the inflamed peritoneum. In similar in vivo experiments the MEL-14 antibody blocks neutrophil homing by 60-70%. These results further support the importance of the MEL-14 antigen in neutrophil extravasation in vivo and indicate that chymotrypsin could be useful in examining the molecular mechanisms involved in extravasation of leukocytes into a variety of diverse tissue sites of inflammation.     

Biochemical analysis and subcellular localization of a neutrophil-specific antigen, PMN-7, involved in the respiratory burst

The adherence of serum-opsonized yeast to neutrophils results in phagocytosis of these particulate stimuli and activation of the respiratory burst. Both events are mediated or modulated in part by the surface receptors for IgG and complement. The link between the binding of complex particulate stimuli to the cell surface, and the triggering of these neutrophil functions, is not completely understood. We have previously described an anti-human neutrophil, murine monoclonal antibody PMN7C3, which specifically inhibits the respiratory burst of neutrophils stimulated with serum-opsonized yeast. In the present study, we show that the antigen recognized by PMN7C3 (PMN7 antigen) is present on a number of neutrophil proteins, including the recently described group of related leukocyte membrane glycoproteins CR3, LFA-1, and p150,95. The PMN-7 antigen differs from other antigens associated with the C3bi receptor complex (MAC 1, MO 1, OKM1, OKM10) in that it is present only on neutrophils among peripheral blood cells. Furthermore, the binding of PMN7C3 to the neutrophil surface inhibits the activation of the respiratory burst by serum opsonized zymosan without affecting phagocytosis of these particulate stimuli. The cross-linking of cell surface PMN7 antigen by multivalent antibody is associated with the capping and internalization of antigen-antibody complexes, and appears to be necessary for the expression of maximum inhibition of opsonized zymosan-triggered respiratory burst activity. PMN7C3 also binds to a group of granule-associated proteins biochemically distinct from CR3, LFA-1, and p150,95. These granule-associated proteins containing PMN7 antigen can be mobilized to the cell surface with secretion. PMN7 antigen-bearing proteins may play a role in modulating the activation of the respiratory burst associated with phagocytosis of serum-opsonize zymosan.     

Leukocyte procoagulant activity in man: an in vitro correlate of delayed-type hypersensitivity

Human mononuclear leukocytes generate cell-bound procoagulant activity (LPCA) after incubation with an antigen (mumps or tuberculin) to which the donor was previously sensitized. An inhibitor of coagulation appears to be liberated into the extracellular culture fluid during incubation of leukocytes with the sensitizing antigen. Removal of this activity before measuring LPCA resulted in a reliable test that correlated directly with delayed skin reactivity. The assay was particularly sensitive in that cells from weakly sensitized donors who reacted only to high doses of tuberculin (100 TU) in the delayed skin tests produced detectable LPCA in vitro. By contrast cells from weakly sensitized donors did not react to PPD in the lymphocyte blast transformation test or the direct macrophage migration inhibition factor test. The LPCA assay correlated closely with the blast transformation and MIF tests in which cells were used from more strongly sensitized donors who reacted in skin tests with lower doses of tuberculin (1 or 10 TU). The assays were antigen-specific in that cells from donors sensitive to mumps antigen but not to tuberculin reacted only to mumps antigen in vitro. The assay was extremely reproducible; cells from individual donors reacted to the same extent over a period of 8 mo). We propose that the assay system reported here represents an improved method for the measurement of cell-mediated immunity in vitro because it requires fewer donor cells, is technically simpler, and is more sensitive than previously described methods.     

Role of T- and B-lymphocytes in the heterogeneity of human cell-mediated reactions to bacterial antigens]

Leucocytes from 30 patients with allergy to tuberculin and bacterial antigens were treated with antithymus (ATS) and anti-immune globulin (AIGS) sera. The leucocyte migration inhibition test (LMIT) was performed with these antigens. ATS abolished the LMIT induced by tuberculin and sometimes by bacterial antigens (staphylococcal, streptococcal etc.). AIGS frequently abolished the LMIT induced by bacterial antigens, but not by tuberculin. In some cases the treatment with any serum abolished the LMIT induced by the antigens, or, on the contrary, it was abolished only by a successive treatment with both sera. The lymphocyte types (T or B) determining the secondary immune response to the same antigen are different in various patients, as well as they differ in the same patients in relation to diverse antigens. Five types of lymphocyte - antigen interrelation in the LMIT have been distinguished.     

A novel cell surface antigen, 4C8, is expressed on human eosinophils

BACKGROUND: A novel monoclonal antibody, anti-4C8, reacted with human peripheral lymphocytes and monocytes but not with neutrophils. In this study, we investigated whether the 4C8 antigen is expressed on human peripheral eosinophils. METHODS: Expression of the 4C8 antigen on eosinophils was analyzed by flow cytometry and molecular analysis of the antigen was performed with eosinophils by Western blotting. RESULTS: Among human peripheral granulocytes, the 4C8 antigen was expressed on CD16-negative cells but not on CD16-positive cells. The 4C8 antigen also appeared to be expressed on eosinophils. To confirm the latter finding, eosinophils were purified from peripheral blood. On flow cytometric analysis, anti-4C8 antibody reacted with purified eosinophils. On Western blotting analysis, anti-4C8 reacted with a single band of 80 kDa in lysates from purified eosinophils. The correlation between the percentage of eosinophils determined by May-Giemsa staining and the percentage of 4C8-positive/CD16-negative cells among granulocytes was good (r = 0.91, P < 0.0001). CONCLUSIONS: Only a few cell surface antigens are available to distinguish human peripheral eosinophils from neutrophils. The novel cell surface antigen, 4C8, is a useful new marker of human eosinophils.     

Prevalence of mycobacterial sensitivity in Montreal children

The prevalence of sensitivity to atypical mycobacteria was found to be low in 2152 Montreal first-grade children tested with tuberculin and one of four atypical antigens randomly allocated. Forty-nine had positive reactions to an atypical antigen. PPD-G produced more reactions than PPD-A, -B or -K. In Greater Montreal there was a higher prevalence of sensitivity to atypical mycobacteria than in the two suburban areas studied. The range of this prevalence, standardized for the type of antigen, was from 0.86 to 2.92% according to region. The highest prevalence of sensitivity to any single atypical antigen, 5.3%, was found for PPD-G in Greater Montreal.Tuberculous infection was found in 1.35% of the children. Small tuberculin reactions (5 to 9 mm to stabilized PPD 5 TU) require clarification by differential tuberculin testing. More of them are caused by M. tuberculosis than by atypical mycobacteria in this particular age group and region.Routine BCG immunization is not indicated for the particular population studied; when given to individuals at risk, its effectiveness should not be impaired by atypical sensitivity at the low level found locally. Future BCG plans require more epidemiologic data.     

Leukocyte-endothelial cell recognition: evidence of a common molecular mechanism shared by neutrophils, lymphocytes, and other leukocytes

The interaction of leukocytes with endothelial cells is intrinsic to the process of leukocyte extravasation, whether during the entry of blood polymorphonuclear leukocytes and monocytes into sites of acute and chronic inflammation, or during the homing of lymphocytes to lymphoid organs. A lymphocyte surface glycoprotein, defined by monoclonal antibody MEL-14, has been described that appears to mediate lymphocyte recognition of postcapillary venules in peripheral lymph nodes, and to control the migration of lymphocytes from the blood into these lymphoid organs. We now report that the antigenic determinant recognized by MEL-14 is present at high levels on other leukocytes as well, including neutrophils, monocytes, and eosinophils; and we demonstrate involvement of the MEL-14 antigen in neutrophil-endothelial cell interactions. MEL-14 immunoprecipitates a neutrophil surface protein of Mr approximately 100,000, similar in m.w. to the 80,000 to 90,000 dalton lymphocyte surface MEL-14 antigen, and it blocks the interaction of neutrophils with endothelial cells in an in vitro model of adhesion to postcapillary venules in lymph node frozen sections. Neutrophil binding to lymph node venules is also inhibited by PPME, a mannose-6-phosphate-rich yeast polysaccharide that is thought to mimic the endothelial cell ligand for the MEL-14-defined lymphocyte receptor. Interestingly, neither MEL-14 nor PPME exhibit a major effect on neutrophil binding to postcapillary venules in Peyer's patches, suggesting that as for lymphocytes, the neutrophil MEL-14 antigen is involved in recognition of tissue-specific endothelial determinants. Finally, we show that MEL-14 inhibits the capacity of neutrophils to migrate from the blood into sites of acute inflammation in the skin. These observations lead us to propose that receptors for tissue-specific endothelial determinants are utilized by neutrophils and lymphocytes and probably other leukocytes during the physiologic process of leukocyte extravasation in vivo.     

Novel role of ICAM3 and LFA-1 in the clearance of apoptotic neutrophils by human macrophages

Apoptotic cells express eat-me signals which are recognized by several receptors mainly on professional phagocytes of the mononuclear phagocyte system. This “engulfment synapse” can define a safe and effective clearance of apoptotic cells in order to maintain tissue homeostasis in the entire body. We show that the expression of four genes related to apoptotic cell clearance is strongly up-regulated in human macrophages 30 min after administration of apoptotic neutrophils. Out of these the significant role of the up-regulated intercellular adhesion molecule 3 (ICAM3) in phagocytosis of apoptotic neutrophils could be demonstrated in macrophages by gene silencing as well as treatment with blocking antibodies. Blocking ICAM3 on the surface of apoptotic neutrophils also resulted in their decreased uptake which confirmed its role as an eat-me signal expressed by apoptotic cells. In macrophages but not in neutrophils silencing and blocking integrin alphaL and beta2 components of lymphocyte function-associated antigen 1 (LFA-1), which can strongly bind ICAM3, resulted in a decreased phagocytosis of apoptotic cells indicating its possible role to recognize ICAM3 on the surface of apoptotic neutrophils. Finally, we report that engulfing portals formed in macrophages during phagocytosis are characterized by accumulation of ICAM3, integrin alphaL and beta2 which show co-localization on the surface of phagocytes. Furthermore, their simultaneous knock-down in macrophages resulted in a marked deficiency in phagocytosis and a slight decrease in the anti-inflammatory effect of apoptotic neutrophils. We propose that ICAM3 and LFA-1 act as recognition receptors in the phagocytosis portals of macrophages for engulfment of apoptotic neutrophils.     

Down-regulation and inactivation of neutral endopeptidase 24.11 (enkephalinase) in human neutrophils

Neutral endopeptidase (EC 3.4.24.11, NEP) is an integral membrane protein of human neutrophils. NEP is identical with the common acute lymphoblastic leukemia antigen (CALLA) of leukemic cells. The expression of NEP on the surface of neutrophils is down-regulated by endocytosis which can be induced by phorbol 12-myristate 13-acetate (PMA) at 37 degrees C. The activity of the enzyme on the surface of intact cells decreases by 76% within 5 min. The activity can be recovered, however, if the cells are lysed within 5 min of the endocytosis. After 30 min, only 32% of the NEP activity is present in the neutrophil lysates. The loss of activity is presumably due to proteolytic inactivation. Diacylglycerol and monoclonal antibody to CALLA/NEP also induce internalization of NEP. PMA induces endocytosis even at 4 degrees C, but NEP is not inactivated at that temperature. The disappearance of NEP activity after adding PMA was inhibited by various agents. Among the most active were the phospholipase inhibitor 4-bromophenacyl bromide and a combination of the serine protease and cathepsin inhibitors, diisopropylfluorophosphate and N-ethylmaleimide. The employment of fluorescent monoclonal antibody confirmed the down-regulation and internalization of NEP antigen on the neutrophils. Since NEP inactivates chemotactic peptides and thereby affects chemotaxis of neutrophils (Painter, R. G., Dukes, R., Sullivan, J., Carter, R., Erd?s, E. G., and Johnson, A. R. (1988) J. Biol. Chem. 263, 9456-9461), the down-regulation of NEP activity on the cell membrane may modulate the function of these cells in inflammation.     

Causes of para-allergy to tuberculin

Experiments on 56 rabbits infected with microorganisms of the genera Mycobacterium, Nocardia and Rhodococcus revealed that in response to the action of different antigens used T and B systems of immunity induced synthesis of antibodies reactive with nonspecific antigens (mycobacterial antigens, tuberculin). In some cases the statistically significant correlation between the dynamics of blast transformation and the specific lysis of lymphocytes in animals with Nocardia and Rhodococcus infections (in comparison with the controls) was determined when P.P.D. tuberculin was used as specific antigen. In the rabbits sera infected by Nocardia and Rhodococcus complement-fixation and hemagglutination antibodies to mycobacterial antigens were detected. These rabbits also exhibited skin reaction to P.P.D. tuberculin. The presence of common group-specific antigens in Nocardia and Rhodococcus, as well as in mycobacteria, determined the capacity of the former to sensitize experimental animals to tuberculin, with should be taken into consideration in making the allergic test to tuberculosis.     

Expression of the CD15 differentiation antigen (3-fucosyl-N-acetyl-lactosamine, LeX) on putative neutrophil adhesion molecules CR3 and NCA-160.

The expression of the carbohydrate antigen 3-fucosyl-N-acetyl-lactosamine (CD15, LeX) on human neutrophil glycoproteins has been studied by immunoprecipitation and immunoblotting by using monoclonal antibody MC2. The antigen is expressed on membrane glycoproteins of approximate molecular mass 165 and 105 kDa. These glycoproteins include the complement receptor and adhesion molecule, CR3, in which the beta-chain (CD18, 105 kDa) shows much greater expression than the alpha-chain (CD11b, 165 kDa). Most of the 165 kDa CD15 antigen is accounted for by expression on the carcinoembryonic antigen (CEA)-related molecule NCA160. Other members of this family, NCA95, NCA90 and NCA55, which are also found in neutrophils, do not express the CD15 antigen. There is a marked increase in the surface expression of CD15, CR3 and the antigen recognized by anti-CEA antibodies upon activation of neutrophils by the chemotactic peptide N-formylmethionyl-leucylphenylalanine.     

The production of mycobacterial antigens by homogeneous culture in a fermentor

Mycobacterium bovis strain BCG, substrain 1173P2, has been grown in homogeneous culture in classical synthetic Sauton medium without supplementary ingredients. The culture conditions are described. The protein release in the culture medium and the tuberculin yield after 2% trichloroacetic acid precipitation were significantly improved. The antigenicity of the tuberculin has been successfully assayed on specifically sensitized guinea-pigs. It is concluded that homogeneous mycobacterium culture in a fermentor using synthetic medium is a suitable method for the large scale production of antigen.     

Competitive antibody binding to soluble CD16B antigen and CD16B antigen on neutrophils in whole blood by flow cytometry

BACKGROUND: Shed receptors from the surface of white blood cells in whole blood have been quantitated using the long and tedious enzyme-linked immunosorbent assay (ELISA) method. A simple rapid flow cytometric method of analysis for shed antigen in the presence of cell-bound antigen can be advantageous. METHODS: Magnetic bead depletion of neutrophils in whole blood with CD16b antibody-conjugated beads as measured by flow cytometric analysis of the remaining cell suspension was inhibited by the presence of soluble CD16b antigen in the blood plasma of normal donors. We describe a competitive binding assay between labeled and unlabeled CD16b antibody for receptors shed from the surface of formed bodies (cells) into solution. Also presented is a new method of obtaining the amount of soluble antigen in a sample. We determine the total unlabeled and labeled ligand concentration at which the fluorescence intensity of the labeled ligand matches the fluorescence intensity in a control run with only the labeled ligand. RESULTS: Normal blood donors showed serum concentration levels of shed CD16b antigen in the range of 1-50 nM as determined by a flow cytometric competitive binding assay. These figures compared favorably with parallel determinations using magnetic bead depletion of targeted neutrophils for washed and unwashed whole blood samples to evaluate the concentration of shed CD16b antigen. CONCLUSIONS: The competitive antibody binding assay for shed and cell-bound CD16b antigen can be applied to similar GPI-linked antigens, for which purified antibody and fluorescent antibody against the same antigenic receptor are available.     

Skin reactivity to mycobacterial antigens in children living in an area of Mycobacterium xenopi endemicity

Intradermal skin tests with a 2TU dose of PPD-RT 23 prepared from M. tuberculosis and 0.1 ug/0.1 ml of PPD-RS 631 from M. xenopi were simultaneously carried out in 378 7-year-old children from two localities in North-Bohemian region's capital Ustí n. Lab., a focus of M. xenopi endemicity repeatedly confirmed since its disclosure in 1980 by positive M. xenopi isolations from humans and public water supply network. A further group 157 children serving as controls was from Prague district 4 where no presence of M. xenopi strains was ever recorded. All of these children had received routine immunization at birth with Czech BCG vaccine. The children from the two endemic localities were found to give a positive 6 mm or greater reaction to M. xenopi mycobacterin in 43.3% and 22.3%, to human tuberculin in 12.8% and 12.6%, respectively. The frequency histogram clearly separated a group of reactors with 8-18 mm indurations from a group of nonreactors showing a skin induration of 4-8 mm. The higher reactivity of this exposed child population was also reflected in a larger proportion of reactions greater to M. xenopi PPD than to human tuberculin antigen: the reactions greater by 1-5 mm accounted, respectively, for 25.1% and 20.6%, reactions greater by 6 mm or more for 23.7% and 15.9%. Among a group of children from Prague district 4, 6.4% had medium-sized and 3.8% large-sized reactions to M. xenopi antigen; the proportion of reactions greater to M. xenopi antigen than to human tuberculin accounted for only 5.1%, reactions greater to tuberculin than to sensitin were here in slight predominance. The evidenced skin sensitization to M. xenopi mycobacterin is suggested to result from the different degrees of exposure to infection by environmental mycobacteria.     

Enhancement of the expression of a rat neutrophil-specific cell surface antigen by activation with phorbol myristate acetate and concanavalin A

A monoclonal antibody against rat neutrophils, RP-1, was produced by hybridizing a mouse myeloma (P3-X63-Ag8.653) with BALB/c mouse spleen cells sensitized with peritoneal neutrophils from Wistar-King-Aptakeman/Hok rats. RP-1 specifically reacted to neutrophils and bone marrow cells from rats of various strains. The expression of an antigen reactive to RP-1 on rat peritoneal neutrophils was enhanced by stimulation with phorbol myristate acetate and concanavalin A. The experimental result that the expression of an antigen reactive with another anti-neutrophil monoclonal antibody was not enhanced by stimulation with phorbol myristate acetate indicates that the antibody-binding capacity of stimulated neutrophils was not nonspecifically enhanced. The enhancement of antigen expression was temperature dependent. A glycolytic inhibitor, 2-deoxy-D-glucose, and an inhibitor of intracellular calcium mobilization, 3,4,5-trimethoxybenzoic acid 8-(diethylamino)-octyl ester hydrochloride, which inhibited hydrogen peroxide release from stimulated neutrophils, did not inhibit enhancement of the expression of an antigen reactive with RP-1.     

In vitro study of macrophages from patients with sarcoidosis

Cultures of leukocytes from patients with sarcoidosis were stimulated with Kveim antigen or PPD-S and the macrophage reactions were compared with those obtained under similar conditions with leukocyte cultures from healthy tuberculin reactors. The non-stimulated control cultures of Kveim-positive sarcoid patients showed signs of macrophage activation, namely an increase in cytoplasmic pyroninophilia in small and enlarged macrophages, together with a higher than normal rate of lymphoblast transformation. In the presence of Kveim antigen this activation was more marked, even in cases where the blast response to the antigen was of low magnitude. Stimulation with tuberculoproteins produced a slight activation of macrophages only in cultures from Kveim reactors who had also shown positive tuberculin reactions.These observations suggest that the study of lymphocyte transformation together with that of macrophage activation will improve the immunocytological assessment of sarcoidosis.     

CD69 molecule in human neutrophils: its expression and role in signal-transducing mechanisms.

The CD69 glycoprotein is an early activation antigen of T and B lymphocytes and it is constitutively expressed on thymocytes and platelets. Here we report its presence on neutrophils and on bone marrow-derived myeloid precursors. Indeed, promyelocytic cells are CD69+ on the cell membrane, while in resting neutrophils this molecule is located inside the cell. However, intracellular CD69 molecules are rapidly mobilized to the cell surface upon activation by PMA or fMLP. This translocation is independent on a new protein synthesis, as it is not inhibited by cycloheximide; furthermore, CD69 molecules are likely stored in a trans-Golgi structure since their expression is not affected by brefeldin A, a drug that blocks molecular trafficking from ER to Golgi vesicles. Immunoprecipitation of CD69 molecules either from activated neutrophils or from bone marrow cells showed that this protein has the same molecular size (28-34 kDa) as observed in platelets, T and B lymphocytes, and thymocytes. This similarity is reflected also in the functional role played by this molecule: in neutrophils as well as in lymphocytes and platelets, CD69 stimulation induced Ca2+ influx through cellular membrane; furthermore, the perturbation of the CD69 antigen on PMA-activated neutrophils enhances the lysozyme release, suggesting a role of this molecule in the regulation of granule exocytosis, probably through a Ca(2+)-dependent mechanism.     

Platelet-associated tissue factor contributes to the collagen-triggered activation of blood coagulation

The extravascular localization of tissue factor (TF), the central initiator of coagulation, is thought to ensure that thrombus formation is prevented in the intact vessel. We observed that during a 5-min stimulation of human blood with collagen (type I), TF antigen appeared on the surface of platelets adhering to leukocytes. The rapidly presented intravascular TF was competent to start the coagulation cascade. The isolated platelets from healthy donors contained appreciable amounts of the TF protein, while no TF antigen was detected in the neutrophils and rapidly isolated monocytes. Direct interactions with the neutrophils and monocytes were apparently necessary to activate the platelet-associated TF. This was most likely mediated by inactivation of tissue factor pathway inhibitor through leukocyte elastase. In summary, the leukocyte-elicited activation of the platelet TF participates in the rapid initiation of coagulation by collagen.