Studies on delayed hypersensitivity pleural exudates in guinea-pigs. II. The interrelationship of monocytic and lymphocytic cells with respect to migration activity.

The in vitro migration activity of monocytic cells and the effect of lymphocytes in the delayed hypersensitivity (DH) reaction induced by intrapleural injection of PPD into FCA-sensitized guinea-pigs was investigated. The in vitro migration of exudate mononuclear cells was lower than that of comparable blood cells, and continued to decrease as the reaction progressed. An inverse relaationship was observed between the migration area and cell volume of mononuclear cells from both blood and exudate. Similar experiments were performed on the reversed passive Arthus (RPA) reaction in the pleural cavity of guinea-pigs. For 6-h exudate, the mononuclear cell migration exceeded that for blood mononuclear cells. However, the migration of mononuclear cells from 18-h exudates was markedly reduced. As with DH, an inverse relationship between the migration area and cell volume was observed with the mononuclear cells of RPA exudates. Following the addition of antigen, the migration of DH exudate mononuclear cells was inhibited whereas the exudate mononuclear cells of the RPA reaction remained unaffected. For DH, the inhibition of migration induced by antigen appeared to be related to the non-adherent cells of 6-h exudates, and both the adherent and non-adherent cells of 18-h exudates.     

Studies on delayed hypersensitivity pleural exudates in guinea pigs. I. Demonstration of substances in the cell-free exudate which cause inhibition of mononuclear cell migration in vitro.

Leucocyte migration inhibitory (MI) activity was investigated in vitro in the delayed hypersensitivity reaction induced by intrapleural injection of PPD into FCA-sensitized guinea-pigs. During the initial reaction (6 h) two types of antigen-dependent MI activity were detected in serum and cell-free exudate. One was of high molecular weight and associated with immunoglobulin and the other was of low molecular weight and appeared to be related to so-called "antigen-dependent MIF". As the reaction progressed (i.e. 12-24 h), two types of antigen-dependent MI activity were revealed in exudate, but not in serum. One was high molecular weight and the other was low molecular weight and thought to be related to so-called "antigen-independent MIF". Similar experiments were performed on the reverse passive Arthus reaction in the pleural cavity of guinea-pigs. A high molecular weight MI activity was detected in 6-h cell-free exudate and was found to be antigen-independent. "So-called MIF" was not found in this reaction.     

Presence and production of migration inhibitory activity in the peritoneal cavity.

The presence of migration inhibitory (MI) activity was investigated in the delayed hypersensitivity (DH) reaction induced by i.p. injection of PPD into FCA-sensitized guinea-pigs. Peritoneal exudates were studied at several times before and after the induction of the DH reaction and a non-immune control inflammation. Lymphokine (LK) fractions were prepared from individual exudates and tested in a conventional macrophage migration inhibition assay at concentrations determined to occur in vivo. The results suggest a continuous local production of MI activity and an augmented production during development of a DH reaction.     

A model for the quantitative study of Arthus (immunologic) hypersensitivity in rats

A model of reverse passive Arthus (RPA) reaction in the pleural cavity of rats is described. The time course of development of exudate and migration of cells has been examined. It has been found to be complement dependent and dominated by polymorphonuclear cells. The reaction reaches a peak around 6 hours after challenge. Cyclic AMP levels have been measured both intracellularly and extracellularly and have been found to persist at high levels after the waning of the reaction.     

Production of leucocyte inhibitory factor (LIF) and macrophage inhibitory factor (MIF) by PHA-stimulated lymphocytes.

Supernatants from human mononuclear cells cultured with PHA inhibited the migration of both human polymorphonuclear leucocytes and guinea-pig peritoneal exudate cells, but not human mononuclear cells. Using ultrafiltration it was shown that these supernatants contained two inhibiting factors, the one with a molecular weight of 15,000-50,000 inhibited only guinea-pig peritoneal exudate cells (MIF), whereas the fraction containing molecules of a size between 50,000 and 75,000 specifically inhibited the migration of polymorphonuclear leucocytes (LIF). The polymorphonuclear leucocyte inhibiting activity was heat labile. It is suggested that the leucocyte migration inhibition test is dependent upon the production of a lymphokine (LIF) which acts specifically on polymorphonuclear leucocytes causing their inhibition of migration.     

Studies on the ability of inflammatory exudate obtained from acute and chronic phases of the inflammatory process to promote leukocyte locomotion in vitro

Using a modified Boyden chamber system, inflammatory exudates taken from the acute carrageenan pleural model and the chronic carrageenan air-pouch model were tested for their ability to promote leukocyte locomotion in vitro. Both exudates were able to induce polymorphonuclear leukocyte (PMN) and mononuclear leukocyte (MN) migration, suggesting that cell-specific chemoattractants are not responsible for the progression of the inflammatory process from the acute to the chronic phase. Checkerboard assays were used to establish whether the observed migration was in response to chemotactic stimulation and/or chemokinetic stimulation. Both acute and chronic exudates were able to induce MN chemotaxis and chemokinesis. Acute exudate was able to induce PMN chemotaxis and chemokinesis, but chronic exudate was only able to induce PMN chemokinesis. This may partly explain the predominance of MNs in chronic inflammation. However, our present in vitro results have failed to demonstrate that cell-specific chemoattractants are responsible for the in vivo observation that migration of polymorphonuclear leukocytes precedes the migration of mononuclear cells.     

The response of the newborn rat to intrapleural dextran: Vascular and cellular aspects

The development of hind paw oedema, in response to dextran injected into the pleural cavity of rats, has been shown to differ with the age of rat studied. It was found that newborn rats were less responsive than adults, with respect to oedema formation induced by dextran. Attempts to suppress the dextran-induced foot oedema using mepyramine and cyproheptadine, were successful only in rats from the adult age group.A semiquantitative analysis was made of the inflammatory cellular exudate produced by the intrapleural injection of dextran into rats of different ages. In contrast to the reaction of adult rats, it was found that the accumulation of polymorphs in the pleura of newborn animals was delayed. The polymorph was the dominant cell type found in exudates induced either by intrapleural saline or dextran, in rats from the 6-hour age group. This contrasted strikingly with comparable exudates induced in adult rats, whose major cell type was the mononuclear cell (after saline), and the polymorph, followed by the mononuclear cell (after dextran).Fewer cells were observed in the reaction of 6-hour-old rats, compared with 7-day-old animals following intrapleural injection of the same volume of a 6-percent solution of dextran.     

Studies on cell motility in inflammation. I. The chemotactic activity of experimental,immunological and non-immunological,inflammatory exudates

The accumulation of leucocytes at the site of inflammation may be brought about by chemotaxis or proliferation in the extravascular tissues. The present paper focuses on the chemotactic properties of different types of experimental inflammatory pleural exudates, using a modified Boyden chamber. The time-course of carrageenan-induced exudate chemotactic activity for polymorphs was maximat at 4 h, thereafter diminishing towards 24 and 48 h. Chemotactic activity for mononuclear cells remained unchanged throughout the 4–48 h time-course. Heating the exudates to 56°C for 1 h partially reduced chemotactic activity. These results correlate well with the migration of polymorph and mononuclear cells into the pleural cavity during carrageenan-induced pleurisy. The potency of polymorph and mononuclear cell chemotactic activity of different exudates was of the following order: carrageenan > calcium pyrophosphate > reverse passive Arthus > dextran. These results are discussed in order to elucidate the differences between the underlying mechanisms responsible for leucocyte accumulation in different types of inflammatory reaction.     

Properties of T cells synthesizing macrophage migration inhibition factor in the H-2 system

It was shown by means of the indirect macrophage migration inhibition test in the H-2 system, using fractionated lymphocytes, that macrophage inhibition factor (MIF) is produced by T but not by B cells. Cells producing MIF are more sensitive to the action of anti--antibodies than T-killer cells. MIF formation by lymph node cells was detected at earlier periods after immunization than the cytotoxic activity of these cells. The results obtained are evidence of differences between the subpopulation of T cells synthesizing MIF and cytotoxic T lymphocytes.Laboratory of Immunochemistry and Diagnosis of Tumors, Oncologic Scientific Center, Academy of Medical Sciences of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR L. M. Shabad.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 85, No. 6, pp. 701–704, June, 1978.     

An assessment of the delayed hypersensitivity reaction to methylated bovine serum albumin in the mouse and its use in the evaluation of drug effects on cell mediated immune reactions

A delayed hypersensitivity (DH) reaction is induced by a sensitizing intradermal injection of methylated bovine serum albumin (MBSA) into the abdomen of mice and a subsequent challenge injection of MBSA into the hind paw. Paw volume increase is measured by mercury plethysmography. The conditions for sensitization have been investigated. Sensitization with a 0.25% MBSA emulsion resulted in a small but significant swelling of the paw following the challenge injection. The magnitude of the footpad response to the challenge injection was increased if the antigen administered in the sensitizing injection was emulsified with Freund's incomplete adjuvant. Incorporation ofMycobacterium butyricum in the emulsion greatly increased the footpad response if added at doses of 0.05 and 1 mg per animal. A higher dose (4 mg), however, resulted in a lower response. The time course of development of the delayed hypersensitivity reaction has been studied. An 8-day interval between sensitization and challenge resulted in a greater delayed hypersensitivity response than a shorter (3-day) or longer (15-, 21-, 28-day) interval. Cyclophosphamide (250 mg kg^–1) administered 3 days prior to the sensitizing injection of MBSA produced a modest enhancement of the DH reaction.On the basis of these studies a protocol for conducting the DH reaction to MBSA was established and the activity of drugs on processes underlying the sensitization phase of the reaction or processes underlying the elicitation phase of the reaction have been examined. Steroid and immunosuppressant drugs were found to inhibit the DH footpad response when dosed during the sensitization whereas several specific-anti-rheumatic and immunoactive compounds were without effect. Indomethacin and sudoxicam inhibited the DH reaction if dosed during the elicitation of the reaction but other non-steroidal anti-inflammatories tested did not significantly reduce the response. The clinically used anti-rheumatic drugsd-penicillamine and levamisole did not inhibit the elicitation phase of the DH response but niridazole at 100 mg kg^–1 did reduce the inflammatory response.This paper has been presented in part as a poster presentation to the British Pharmacological Society, 4–6 January 1978.     

Inflammatory mechanisms in the newborn

Both the vascular and cellular aspects of inflammation were studied in rats aged from 6 hours to 2 months. The onset of a vascular permeability reaction was found to vary according to the age of rat and type of permeability agent injected.Induction of acute inflammations (turpentine pleurisy, 48/80 and dextran-induced foot oedema) in newborn rats revealed a marked reduction of exudate/oedema formation compared with adult rats. These observations were related to a lack of histamine and 5-HT-mediated increased vascular permeability during the inflammatory reactions of newborn rats.Qualitative differences were observed between the acute inflammatory pleural cell exudates of newborn and adult rats after intra-pleural injection of dextran. In contrast to adults, polymorphs were observed to dominate the newborn reaction, and the peak accumulation of these cells was delayed. The mononuclear cells of 4-day lesions induced by sub-cutaneous implantation of glass coverslips showed a higher rate of mitosis in newborn animals, compared with adults.Ultrastructural studies of the mononuclear cells of peritoneal exudates induced by carrageenan indicated that those of newborn rats contained fewer lysosomes than adults. The Golgi apparatus of newborn mononuclear cells was observed to be poorly-developed compared with adult mononuclear cells.     

Inhibition of Migration of Mouse Macrophages by Tuberculin-Sensitive Mouse Lymphocytes and by Mouse Migration Inhibitory Factor

The guinea pig migration inhibition technique, an accepted in vitro correlate of delayed hypersensitivity, has been adapted to a murine system. Peritoneal exudate cells from CF-1 mice vaccinated with viable cells of the H37Ra strain of Mycobacterium tuberculosis were inhibited in vitro by purified protein derivative (PPD) or whole H37Ra microorganisms. Peritoneal exudate cells from the inbred C57Bl/6 mice immunized with H37Ra cells also were inhibited in vitro by PPD or whole H37Ra microorganisms. Migration inhibitory factor (MIF) was produced by splenic lymphocytes from the H37Ra-immunized C57Bl/6 mice when incubated with either antigen. Intravenous injection of PPD or viable H37Ra organisms into H37Ra mice resulted in MIF production in vitro by splenic lymphocytes without further antigenic stimulation. Peritoneal exudate cells from nonimmunized C57Bl/6 mice and supernatant fluids from cultures of lymphocytes from nonimmunized C57Bl/6 mice were not inhibited in the presence of antigen. The production of MIF by splenic lymphocytes from immunized C57Bl/6 mice depended upon the conditions under which the lymphocytes were cultured, the time of exposure to antigen (3 days), the use of a higher concentration of PPD for stimulation of lymphocytes than that required for guinea pig cells, and also the use of cells from a highly inbred mouse strain.     

Development of the cellular immune response to tuberculin in mice of different genotypes

Inbred CBA and C57BL mice and (CBA×C57BL)F₁ hybrids were immunized intraperitoneally with Freund's complete adjuvant and the production of macrophagal migration inhibiting factor (MIF) by lymphocytes in different situations was investigated. Lymphocytes were activated to produce MIF in a certain order: cells of the peritoneal exudate first, then lymph node cells and, finally, spleen cells. Maximal MIF production by lymphocytes of the peritoneal exudate was obtained in C57BL mice, and it occurred earlier (on the 3rd day after immunization). Increased spontaneous migration of macrophages also was observed after immunization and it was more marked in the CBA mice.Department of Immunology, N. I. Pirogov Second Moscow Medical Institute. (Presented by Academician of the Academy of Medical Sciences of the USSR Yu. V. Lopukhin.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 82, No. 8, pp. 972–974, August, 1976.     

Characterization of phospholipase A2 activity in rat RPAR-induced pleurisy

At 10 min, 2 h and 4 h after induction of a reverse passive Arthus reaction (RPAR) in the pleural cavity of rats, pleural exudate was collected and analyzedin vitro for phospholipase A₂ (PLA₂) acyl hydrolytic activity. Inasmuch as exudate PLA₂ was inhibited by addition of EDTA, the acyl hydrolytic activity appeared calcium-dependent. However, addition of exogenous calcium to the exudate had only a minor effect on the activity. Maximum hydrolysis in all exudates was observed over a pH range of 6–9 with a neutral optimum. The 4 h exudate PLA₂ activity was almost fully inhibited by para-bromophenacyl bromide (pBPB), whereas the acyl hydrolytic activity in the 10 min and 2 h exudates was reduced by 44% and 46%, respectively, by 200 M pBPB. Although the PLA₂ in the 4 h exudate displayed an increased sensitivity to high exogenous calcium concentrations and to pBPB inactiontion, the basic properties of the exudate PLA₂ at the three time points appeared similar.     

Leukocyte migration inhibition activity of nonimmune acute inflammatory pleural exudate

Material with leukocyte migration inhibition (LMI) activity has been demonstrated in various types of nonimmunologically induced acute pleural inflammatory exudates. This activity is present in inflammatory cell-free exudate and appears to involve the deposition of fibrin around the migrating leukocyte, resulting in cell-trapping. This is supported by the fact that removal or inhibition of fibrin formation leads to loss of exudate LMI activity. Both fibrinogen and complement as well as vitamin K-dependent clotting factors appear to be required for LMI activity. The mechanism involved in the LMI reaction and its significance in nonimmune and cell-mediated immune inflammation are discussed.     

Macrophage Migration Inhibition Studies with Cells from Mice Vaccinated with Cell Walls of Mycobacterium bovis BCG: Characterization of the Experimental System

The macrophage migration inhibition test was applied to the study of delayed hypersensitivity in mice vaccinated intravenously with oil-treated cell walls of Mycobacterium bovis BCG. Migration inhibition of peritoneal exudate cells from sensitized mice was demonstrated directly upon incubation of the cells with purified protein derivative, but indicator cells such as normal peritoneal cells had to be included to demonstrate migration and migration inhibition with sensitized lung cells. Inhibition of migration induced by mouse cells was greatest 3 to 4 weeks after sensitization but was still considerable after 11 weeks. The migration inhibitory factor (MIF) was not detected in cells freshly isolated from sensitized mice but was released into the supernatant fluid when cells were incubated with purified protein derivative for 24 hr at 37 C in a tissue culture system. Production of MIF was inhibited by actinomycin D and puromycin. MIF was nondialyzable, resistant to heating at 56 C for 1 hr, and of a lower molecular weight than mouse gamma globulin. All data indicated that migration inhibition induced by cells from cell wall-vaccinated mice was very similar to that caused by guinea pig lymphocytes.     

Integrin α6 expression in human prostate carcinoma cells is associated with a migratory and invasive phenotypein vitro andin vivo

Cell adhesion and migration are important features in tumor invasion, being mediated in part by integrins (extracellular matrix receptors). Integrins are significantly decreased in human prostate cancer. An exception is 6 integrin (laminin receptor) which persists during prostate tumor progression. We have selected high (DU-H) and low (DU-L) expressors of 6 integrin from a human prostate tumor cell line, DU145, to assess experimentally the importance of 6 integrin in tumor invasion. DU-H cells exhibited a four-fold increased expression of 6 integrin on the surface compared to DU-L cells. Both cell types contained similar amounts of 3 and 5 integrin. The DU-H cells contained 6 subunits complexed with both the 1 and 4 subunits whereas DU-L cells contained 6 complexed only with 4. DU-H cells were three times more mobile on laminin as compared to DU-L, but adhered similarly on laminin. Adhesion and migration were inhibited with anti-6 antibody. Each subline was injected intraperitoneally into SCID mice to test its invasive potential. Results showed greater invasion of DU-H compared to DU-L cells, with increased expression of a6 integrin on the tumor at the areas of invasion. These data suggest that 6 integrin expression is advantageous for prostate tumor cell invasion.     

Effect of acute nonimmune inflammation on locomotion of exudate and blood rabbit neutrophils

The random and directed locomotion of rabbit polymorphonuclear neutrophils (PMNs) were investigated in vitro by the agarose technique. PMNs were either recruited in serum-elicited pleural exudates or isolated from blood collected before or after pleurisy development. Directed PMN migration was assessed in the presence ofN-formyl-methionyl-leucyl-phenylalanine (FMLP), isologous rabbit serum (IRS) or cell-free exudates as chemotactic stimuli. Neutrophils collected from blood before pleurisy induction or 4 h afterwards displayed similar random migration, and similar migration oriented by FMLP, IRS, or cell-free exudates. However, both random and FMLP-induced migration were greater for exudate than blood PMNs. In the presence of IRS or cell-free exudate as chemoattractants, exudate and blood PMNs migrated over similar distances. These results suggest that exudation does not deactivate elicited PMNs but alters their locomotion responses according to the stimulus applied.     

Migration inhibitory factors and macrophage differentiation

Summary It has been described before that only certain types of macrophages are capable to respond to lymphokines and that only certain macrophage phenotypes were able to migrate and to respond to migration inhibitory factors (MIF). With respect to the dissociation of MIF activities from a series of other biological activities, and with regard to the phenotype-associated response of macrophages to MIF it was asked: What are the characteristics of the MIF-responsive macrophage phenotype and what are the functional changes induced by MIF on macrophages in addition to inhibition of random migration? Bone marrow-derived macrophages on day 6 of culture were separated by hypotonic Percoll density gradient centrifugation into three distinct bands and analyzed for a variety of functions. It was found that migrating and MIF-responsive macrophages accumulate at a certain density. These macrophages were further characterized by monoclonal antibodies generated against murine macrophage phenotypes. One marker was found to be preferentially expressed by MIF-responsive macrophages. In order to study the inducibility of MIF responsiveness, bone marrow-derived macrophages on day 16 of culture which were poorly migrating and did not respond to MIF were induced to proliferate by the addition of L cell-conditioned medium. After proliferation had subsided, MIF sensitivity was restored. The effects of MIFs other than migration inhibition, on a number of functions which had been mapped within the cell cycle, were investigated. It was found that MIF acts anti-proliferative on young, cycling macrophages. Non-cycling, mature macrophages were shifted to a state characterized by a decreased expression of transglutaminase and plasminogen activator and an increase of certain phenotypic surface markers. It is concluded that MIFs are differentiation-inducing signals, acting on the generation of macrophages from precursors but also in the recruitment of terminally differentiated macrophages to inflammatory type of macrophages which are functional in the induction of immune responses.     

Studies on cell motility in inflammation. II. The in vivo effect of anti-inflammatory and anti-rheumatic drugs on chemotaxis in vitro

Two systems were used to test the effect of anti-inflammatory and anti-rheumatic drugs on chemotactic activity of cell-free exudates and also on the chemotactic responsiveness of exudate leucocytes. (1) Inflammatory cell-free exudates from treated rats were tested for their chemotactic activity on exudate leucocytes from untreated rats. (2) Polymorph and mononuclear cells from treated rats were tested for their responsiveness to the chemotactic activity of cell-free exudates from untreated rats. Levamisole, coumarin and D-penicillamine were ineffective in (1) and (2). Colchicine reduced chemotaxis of polymorphs in both systems (1) and (2), whereas no effect was observed on mononuclears. Naproxen was more effective in reducing the chemotaxis of polymorphs compared with mononuclears in systems (1) and (2). In contrast, indomethacin and dexamethasone reduced the chemotaxis of both polymorph and mononuclear cells in systems (1) and (2). Of the drugs tested dexamethasone exhibited the highest potency. These results emphasize the necessity for studying both cellular and humoral factors in the evaluation of the action of anti-inflammatory drugs on chemotaxis.