Immunologic and nonimmunologic activation of macrophages.

Cells of the mononuclear phagocyte system take on different morphological features and appear to have different functions in immunological and nonimmunological granulomas. In immunological granulomas the appearance is of epitheliod cells. Epitheliod cells are of 2 types. In borderline tuberculoid leprosy granulomas, the appearance is of activated macrophages. Similar activation can be induced by lymphokine which increases respiratory enzyme activity. Other types of epithelioid cell are found in experimental zirconium granulomas. These cells contained rough endoplasmic reticulum, indicating that they may play a secretory role. It is suggested that these cells could play a part in stimulating fibrosis. Other substances that produce nonimmunological granulomas, such as aluminum containing compounds, are directly toxic to macrophages in vitro resulting in the rapid release of cytoplasmic enzymes. These compounds also activate complement, causing C3 conversion and anaphylatoxin production. C3 conversion may be through pathways other than the classical and alternative pathways and does not occur in the absence of plasminogen. Mycobacteria can activate complement through the alternative pathway, suggesting a mechanism for granuloma formation in lepromatous leprosy. Loss of C3 membrane receptors from macrophages in lepromatous leprosy could be produced by feeding peritoneal exudate macrophages with mycobacteria in vitro. This was not just due to phagocytosis, as a similar receptor loss was not obtained when the cells were fed latex particles or zymosan. Epithelioid cells in tuberculoid leprosy and sarcoidosis lose Fc membrane receptors but retain C3 receptors. Thus epithelioid cells can be readily distinguished from other cells of the mononuclear phagocyte series.

Sensitization with Cr, Ni and Zr salts and allergic type granuloma formation in the guinea pig.

Guinea pigs were sensitized to potassium dichromate, nickel sulfate and sodium zirconium lactate by three methods of immunization in Freund's complete adjuvant: Polak, split adjuvant, and maximization (modified Magnusson and Kligman). These were followed after 2 weeks by weekly intradermal injections of 25 microng of the metal salt. Delayed hypersensitivity-like reactions developed 3 to 12 weeks after initial injection. Reactivity, although strong with an increase in thickness of over 0.7 mm and/or diameter of erythema of over 8 X 8 mm, was frequently transient, the animal losing reactivity on subsequent skin test or after 2 or 3 skin tests. In two-thirds of the experiments using sodium zirconium lactate, delayed hypersensitivity-like reactions at 24 hr developed into nodular lesions which reached peak intensity at 8 days and histologically contained histiocytes with an epithelioid cell appearance and giant cells. In some experiments, sodium zirconium lactate-sensitive animals showed cross reactivity with potassium dichromate, but not with nickel sulfate.

Predictive value of assessment of lymph node weight and T-lymphocyte proliferation in contact sensitivity in acrylates.

An assessment of T-lymphocyte proliferation and lymph node weight is proposed as a predictive test for contact sensitizers of industrial origin. Data are presented showing increased T-lymphocyte proliferation following epicutaneous application of a variety of industrially important acrylate-like chemicals which appear to correlate well with their ability to sensitize in the guinea pig. These data were compared with those obtained after application of 2,4-dinitro-1-fluorobenzene (DNFB) a strong sensitizer, and 2,4-dinitrothiocyanatebenzene (DNTB) a nonsensitizer when given epicutaneously. It is suggested that this quantitative approach, in parallel with a simple one-dose immunization, may provide a better picture of sensitization potential than the longer multidose immunizations currently in use.

A comparison of the conjugation of DNTB and other dinitrobenzenes with free protein radicals and their ability to sensitize or tolerize.

Of several dinitrobenzenes tested, 2,4-dinitrothiocyanatebenzene (DNTB) was found to be the only one that did not induce contact sensitivity when applied to the guinea pig ear epicutaneously, but when applied epicutaneously it induced tolerance to 2,4-dinitrofluorobenzene (DNFB). The manner in which DNFB, DNTB, and other dinitrobenzene compounds conjugated in vitro to soluble proteins, at physiologic pH, was examined. By measuring the free amino and sulfydryl radicals in the protein before and after conjugation, it was possible to determine to which groups the hapten was bound. It was found that although all the haptens bound to the free sulfydryl groups, DNTB was the only one that did not bind to amino groups. It is suggested that to be an epicutaenous tolerizer, as opposed to sensitizer, a hapten should bind to sulfydryl groups exclusively. It is hoped that a search for agents binding in a similar manner will reveal epicutaneous tolerizers for important industrial sensitizers.

The effect of photochemotherapy (PUVA) on cell mediated immunity in the guinea pig.

Guinea pigs were injected intraperitoneally with 8-methoxypsoralen (8-MOP) and afterwards the shaved right flank was irradiated with UV-A. This treatment was performed daily for 14 days. Seven days after treatment started they were immunized with ovalbumin in Freund's complete adjuvant and 7 days later skin tested with ovalbumin on the unirradiated left flank. Photochemotherapy (8-MOP plus UV-A) did not alter the total white blood cells or the proportion of lymphocytes or granulocytes. However, this treatment significantly depressed the delayed hypersensitivity reaction to ovalbumin; nonspecific inflammation, induced by intradermal turpentine, was not altered. In addition, treatment with 8-MOP alone suppressed the skin reaction to ovalbumin, but not to the same extent as treatment with 8-MOP and UV-A. 8-MOP plus UV-A had no effect on macrophage migration inhibition factor but it did significantly depress mitogen and antigen specific lymphocyte transformation.

Prostaglandin levels in cerebrospinal fluid from multiple sclerosis patients in remission and relapse.

Radioimmunoassay (RIA) techniques have been employed to determine prostaglandin (PG) levels in the cerebrospinal fluid (CSF) from multiple sclerosis (MS) patients in remission and relapse and in subjects with other neurological diseases (OND). PGE and PGF2 alpha concentrations in spinal fluid from MS patients in relapse were significantly lower than values estimated during remission and in individuals with OND of the central nervous system (CNS). These observations are discussed in relation to the clinical state of patients with demyelinating disease together with a consideration of the concept that disordered immune mechanisms contribute a central role in the pathogenesis of MS.

A study of the prostaglandin and thromboxane content of the central nervous tissues with the development of chronic relapsing allergic encephalomyelitis.

Levels of PGE, PGF2 alpha, 6-oxo-PGF1 alpha and thromboxane (TXB2) in spinal cords and cerebellums of guinea pigs at different stages of chronic relapsing allergic encephalomyelitis (CREAE) were compared with those in Freund's adjuvant-treated, age-matched controls. PGE and TXB2 levels were found to be increased in spinal cords during acute and relapse phases of the disease. The number of lesions in the spinal cord was similarly increased in acute and relapse stages. There was, however, no similar correlation between number of lesions and eicosanoid levels in the cerebellum with the clinical stages of the disease based on hind limb paralysis. In the acute phase and remission lesion numbers were low, and high levels, similar to those found in the spinal cord, were only found in the relapse phase. Eicosanoid levels were high in the acute phase and remission, and generally low in relapse. The spinal cord levels of eicosanoids in remission and relapse correlated well with previous data obtained from the CSF of patients with multiple sclerosis.

Inhibition of chronic relapsing experimental allergic encephalomyelitis in the Biozzi AB/H mouse.

Chronic relapsing experimental allergic encephalomyelitis (CREAE) can be reproducibly induced in Biozzi AB/H mice following injection of spinal cord homogenate (SCH) emulsified in complete Freund's adjuvant (CFA). Active clinical disease is associated with mononuclear cell infiltration of the central nervous system (CNS), mainly the spinal cord. Whole brain homogenate (BH), however, failed to induce clinical or histological disease. In contrast, substituting sciatic nerve homogenate in the inoculum induced experimental allergic neuritis (EAN). Clinical disease was manifest earlier (13.1 +/- 0.3 days) than CREAE (16.2 +/- 1.4) and was accompanied by mononuclear infiltration of the peripheral nervous system (PNS). In comparison to CREAE induction, pretreating mice with SCH or BH in incomplete Freund's adjuvant (IFA) suppressed the development of SCH-induced disease. The BH was more tolerogenic than the SCH and this hyporesponsiveness was CNS antigen-specific as PNS tissue failed to inhibit the course of CREAE. Tolerance induced by pretreatment with SCH or BH in IFA was reversed by a single injection of 200 mg/kg cyclophosphamide, 2 days prior to CREAE induction. This suggests that IFA-induced hyporesponsiveness is actively regulated, possibly via the action of suppressor cells. In addition, treatment with neuroantigens in IFA appears to be mainly afferent acting as it serves to prevent initial disease induction. This treatment after immunization for CREAE, however, fails to prevent disease progression. Furthermore, treatment with CNS antigens emulsified in IFA during the post-acute remission stage appeared to synchronize and induce (32 +/- 1 days) the onset of clinical relapse, compared with untreated controls (41 +/- 5 days). This indicates that such IFA treatment has minimal value in controlling an ongoing immune disease of the CNS.

The modulation of class II histocompatibility antigens and 'activated' macrophage determinant in the spinal cord during the development of chronic relapsing experimental allergic encephalomyelitis (CREAE) in the guinea pig--relevance to the induction of remission?

Cryostat sections of spinal cord of guinea pigs with chronic relapsing experimental allergic encephalomyelitis (CREAE) were stained with monoclonal antibodies recognising a Strain 13-specific Ia epitope, a non-strain-specific Ia antigen and an 'activated' macrophage determinant. It was found that both Ia antigens and the 'activated' macrophage determinant, observed on infiltrating cells within both perivascular and meningeal compartments, appeared to be modulated during the course of CREAE. This correlated with the neurological symptoms of the disease. Blood vessels and 'glial' cells expressed both Ia determinants. 'Glial' cells also expressed the 'activated' macrophage antigen. These antigens were modulated with the course of the disease.

An immunoelectron microscopical study of the expression of class II MHC and a T lymphocyte surface marker during chronic relapsing experimental allergic encephalomyelitis.

Immunoelectron microscopy using antibodies recognising Class II MHC antigens and a pan T cell marker was employed to study sections of spinal cord from guinea pigs with chronic relapsing experimental allergic encephalomyelitis (CREAE). It was found that endothelial cells expressed Class II antigens on their luminal surface throughout the course of the disease and that lymphocytes were adherent to these surfaces. In the parenchyma lymphocytes, macrophages and possibly microglia expressed Class II antigens suggesting that they might also be involved in antigen presentation. The different distribution of T cells seen in the individual lesions during the relapse phase may correlate with their respective natural histories.

A quantitative immunocytochemical study of the infiltrating lymphocytes in the spinal cord of guinea pigs with chronic relapsing experimental allergic encephalomyelitis.

The production and characterization of an anti-guinea pig B cell monoclonal antibody is described. Immunocytochemical techniques using this antibody and others recognizing a Pan T cell antigen and T cell subsets were employed to study frozen sections of spinal cord from guinea pigs with chronic relapsing experimental allergic encephalomyelitis. T and B cells were found in both perivascular lesions and the central nervous system parenchyma, with the major T cell infiltration occurring by the end of the acute phase of disease. The distribution of T cell subsets suggests a phenotypic selectivity in favour of the transport of CT6 (putative CD8)+ve cells across the blood-brain barrier.

An immunoelectron microscopical study of the expression of class II major histocompatibility complex during chronic relapsing experimental allergic encephalomyelitis in Biozzi AB/H mice.

Immunoelectron microscopical techniques have been used to study class II major histocompatibility complex (MHC) expression by cells in the spinal cords of Biozzi AB/H mice with chronic relapsing experimental allergic encephalomyelitis. Throughout the course of disease both astrocytes and endothelia failed to express significant levels of class II MHC antigens. The major central nervous system resident cell types found to express class II MHC antigens were the perivascular microglia, with infiltrating macrophages and some lymphocytes being strongly positive.

Endothelial cell expression of the intercellular adhesion molecule-1 (ICAM-1) in the central nervous system of guinea pigs during acute and chronic relapsing experimental allergic encephalomyelitis.

This study investigated the expression of intercellular adhesion molecule-1 (ICAM-1; CD54) by cells of the central nervous system (CNS) during acute experimental allergic encephalomyelitis (EAE) and chronic relapsing EAE (CREAE). In the CNS of normal guinea pigs, only a few endothelial cells expressed detectable levels of ICAM-1, whereas during the active phases of the disease ICAM-1 was present on cells of the perivascular infiltrate and the endothelia of both lesion- and non-lesion-associated blood vessels. In addition, cultured cerebrovascular endothelia maintained in 'standard' culture medium did not express ICAM-1, but they could be induced to express this antigen on incubation in a lymphocyte-conditioned medium. These findings suggest that the induction of ICAM-1 on CNS endothelia may be important in antigen presentation or in promoting lymphocyte extravasation across the blood-brain barrier in inflammatory disorders of the CNS.

The localisation of fibrin, fibronectin and class II major histocompatibility complex antigen in the spinal cord in chronic relapsing experimental allergic encephalomyelitis.

Light and electron immunocytochemistry using antibodies recognising a class II major histocompatibility complex antigen, fibrin, fibronectin, albumin and factor VIII related antigen has been used to stain sections of spinal cord from normal guinea pigs and those with chronic relapsing experimental allergic encephalomyelitis (CREAE). It was found that class II MHC antigens, fibrin and fibronectin were present in normal blood vessels and at high levels in lesions from animals at all stages of the disease. The possible immunological roles of these antigens suggest their participation in the initiation and maintenance of disease state.

An immunoelectronmicroscopical study of the expression of major histocompatibility complex (MHC) class II antigens in guinea pig sciatic nerves following induction of intraneural mycobacterial granulomas.

A guinea pig model of nerve damage in leprosy has been used to investigate the expression of major histocompatibility complex (MHC) class II antigens in granulomatous lesions in nerves. Using an immunoelectronmicroscopical technique, infiltrating mononuclear cells and endoneural fibroblast-like cells are shown to be class II-positive in the experimental neural lesions. Schwann cells are not class II-positive under these conditions, although at the light microscope level Schwann cell-like cells appear to be positively stained. This illustrates the value of immunoelectronmicroscopy in the investigation of cell surface proteins in situ as compared with conventional light immunohistochemistry.

Control of immune-mediated disease of the central nervous system with monoclonal (CD4-specific) antibodies.

Chronic relapsing experimental allergic encephalomyelitis (CREAE) was induced in Biozzi AB/H (H-2dq1) mice by active sensitization with spinal cord antigens. A single i.p. injection of CD8-depleting (YTS169.4) monoclonal antibody (mAb) failed to affect the clinical course of CREAE when administered prior to and during the onset of both the initial clinical and subsequent relapse phase of the disease. By contrast similar treatment with both CD4-depleting (YTS191.1) or CD4-blocking/non-depleting (YTS177.9) mAb significantly inhibited disease progression. Treatment shortly before the anticipated onset of clinical EAE prevented the subsequent development of disease, although disease could be provoked following antigen-rechallenge. In contrast, treatment with these antibodies during post-acute remission phase mainly served to delay the incidence of relapse. This suggests that, unless tolerance can be re-induced, treatment of ongoing neuroimmunological disease will require 'pulse' therapy and thus potentiate the problems of long-term immunosuppresion. Despite the findings that CD4-specific antibodies can rapidly reverse overt clinical disease shortly after the onset of disease exacerbation, once neurological dysfunction becomes established anti-CD4 treatment fails to improve the animals clinically, possibly due to the inability to rapidly reverse established demyelination. Although this study does not exclude the potential central action of the injected mAb, the failure to significantly dissociate therapeutic benefit between mAb administered directly into the CNS and that given systemically suggests that a major action of these agents is probably by selectively removing T cells in the peripheral T cell pool.

Induction of chronic relapsing experimental allergic encephalomyelitis in Biozzi mice.

Experimental allergic encephalomyelitis (EAE) was induced in Biozzi AB/H (antibody high) mice by sensitization with spinal cord homogenate in adjuvant. Biozzi AB/H mice were highly susceptible to EAE induction and followed a chronic relapsing pattern of disease. Disease episodes were characterized by mononuclear infiltration of the central nervous system, with demyelination being particularly evident in relapse. The cellular infiltrates, which were associated with immunoglobulin deposition, consisted of macrophages and primarily CD4-positive T lymphocytes. However, similarly treated Biozzi AB/L (antibody low) mice were markedly less susceptible to EAE induction than AB/H mice. Thus, Biozzi mice should prove valuable for the study of chronic relapsing EAE.

Identification and quantitation of the expression of T cell surface markers during the development of chronic relapsing experimental allergic encephalomyelitis (CREAE) in the guinea pig.

As there were discrepancies in previous data on the T cell nature of cells infiltrating the meninges at all stages of chronic relapsing experimental allergic encephalomyelitis (CREAE), experiments have been performed using a further monoclonal antibody (Mab) recognizing total T cell populations and the classic E rosetting technique. Cytospins were prepared of the meningeal inflammatory cells obtained by washing the brains of these animals, and stained by indirect immunoperoxidase. It was found that the T cell, as defined by both E rosetting and staining with the Mab CT5, is the major cell type found in the meninges during the development of CREAE. However, the staining with the Mab CT7, which recognizes a functionally relevant antigen, showed that there is a discrepancy between the numbers of lymphocytes stained compared to the results with CT5 and E rosettes. Furthermore, the antigen recognized by CT7 appeared to be modulated during the disease. The possible functional relevance and its relation to clinical remission and relapse are discussed.

Therapy of chronic relapsing experimental allergic encephalomyelitis and the role of the blood-brain barrier: elucidation by the action of Brequinar sodium.

The immunosuppressive effect of the novel 4-quinoline carboxylic acid derivative Brequinar sodium on the chronic relapsing experimental allergic encephalomyelitis CREAE model in the Biozzi AB/H mouse was investigated. Although Brequinar sodium actively inhibited peripheral immune responses, it showed a limited potential to control an ongoing disease of the central nervous system (CNS). Doses of 25 mg/kg inhibited in vivo induced proliferative response and prevented EAE when treated from day 9 post-inoculation (p.i.). However, when administered from day 12 p.i. or during the post-acute remission phase-limited effects on the course of disease were observed. By comparison, treatment with a single high dose of cyclophosphamide (200 mg/kg) at these time points was significantly effective in controlling disease. As a possible explanation of the observed results it is suggested that for a compound to be effective in treating an ongoing immune response in the CNS, it must be capable of crossing the blood-brain barrier and act on the disease-inducing cells activated within the CNS. This hypothesis is supported by the finding that intracerebral injections of Brequinar sodium on day 12 p.i. significantly inhibited disease progression. This suggests that strategies aimed at controlling immune-mediated disease of the CNS require therapeutic doses of the compounds to be delivered into the CNS.

The origin, morphology, and function of epithelioid cells.

Epithelioid cells are cells of the mononuclear phagocyte system found in certain granulomas mainly associated with intense immunological activity. These cells show little phagocytic activity. In certain experimental granulomas such as those produced in guinea pigs sensitive to zirconium, and at sites of intense inflammatory reaction in man, they may contain varying amounts of rough endoplasmic reticulum ("secretory" epithelioid cells). In other situations such as tuberculoid leprosy and in some cases of sarcoidosis they may have the appearance of activated macrophages or take on a multivesicular appearance ("vesicular" epithelioid cells). It is suggested that "vesicular epithelioid cells could develop from "secretory" epithelioid cells by a process of degeneration. In studies comparing granulomas induced in lymph nodes draining the site of intradermal injection of mycobacteria, epithelioid cell granulomas were produced with BCG vaccine, whereas, the granulomas induced by Mycobacterium leprae contained undifferentiated macrophages that contained phagocytosed organisms. The BCG granulomas were in addition characterised by fibroblast infiltration, the presence of collagen and resolution by fibrosis. M. leprae granulomas showed little evidence of fibroblastic activity. Biochemical studies confirmed that BCG granulomas formed new collagen in vitro, whereas this did not take place with M. leprae granulomas. It is suggested that epithelioid cells could play an important role in fibrosis possibly by the secretion of a fibroblast activating factor.