A.J. Suckling

The effect of Freund's adjuvants on blood-cerebrospinal fluid barrier permeability☆

In guinea pigs with experimental allergic encephalomyelitis induced by spinal cord homogenate--complete Freunds adjuvant (CFA) emulsions an increase in the albumin permeability of the blood--cerebrospinal fluid barrier occurred from day 10 post-inoculation (p.i.) onward. In animals inoculated with CFA alone an increased albumin permeability was also demonstrated but only between days 5 and 10 after inoculation; by day 14-16 p.i. the barrier permeability had returned to control values. A similar change was seen in animals inoculated with incomplete Freunds adjuvant (IFA) only. However, both CFA and CFA-cord induced a strong humoral immune response which was not seen in animals inoculated with IFA alone. These results may have important consequences for the understanding of the development of inflammatory diseases of the central nervous system.

Humoral immunity in chronic relapsing experimental autoimmune encephalomyelitis: The major oligoclonal IgG bands are antibodies to mycobacteria☆

Abstract Chronic relapsing experimental autoimmune encephalomyelitis (CREAE) was induced in strain 13 guinea pigs by inoculation with homologous spinal cord (SC) in Freunds adjuvant containing heat-killed Mycobacteria tuberculosis (CFA). Serum and cerebrospinal fluid (CSF) were collected from animals killed during the various clinical phases of the disease and analysed to obtain data on humoral immunity in CREAE, a putative animal model for the human demyelinating disease, multiple sclerosis (MS). Inoculation with either SC/CFA CFA-alone evoked a 2–3-fold increase in serum IgG levels which persisted for at least 10 months. Only in chronic relapse (no remission for >2 months) were IgG albumin ratios in CSF samples significantly greater than those in autologous serum samples. Isoelectric focusing revealed discrete oligoclonal bands of IgG in sera and CSF from both CFA controls and animals in all post-acute phases of CREAE. Autologous serum and CSF samples showed essentially identical IgG spectra. All of the oligoclonal IgG bands in CFA sera and most of those in CREAE sera were removed by incubation with M. tuberculosis before IEF. Although IgG antibodies to antigens of spinal cord homogenate and to CNS polypeptides (predominantly myelin basic protein) were detected in sera from some CREAE animals they did not constitute any of the major IgG bands observed on IEF and were present at concentrations much lower than those to mycobacterial antigens which amounted to 10% of the total serum IgG. The quantity of antibodies to mycobacterial or CNS-antigens and the intensity of the oligoclonal IgG bands in serum samples did not seem related to the clinical status of the guinea pigs. It is suggested that, by analogy, the major oligoclonal IgG bands observed on IEF of CSF from MS patients may represent antibody to infectious agents which act as immunological adjuvants in the human disease.

Identification of a common idiotype on myelin oligodendrocyte glycoprotein-specific autoantibodies in chronic relapsing experimental allergic encephalomyelitis

The myelin/oligodendrocyte glycoprotein (MOG) is a target antigen for autoantibody-mediated demyelination in chronic relapsing experimental allergic encephalomyelitis (CREAE) in the Strain 13 guinea pig. Anti-idiotypic antibodies directed against a demyelinating MOG-specific monoclonal antibody (8-18C5) have identified a cross-reactive idiotype in 10/10 CREAE sera. In nine of these sera inhibition studies demonstrated that this idiotype was at or in close proximity to the combination site of guinea pig anti-MOG autoantibodies. This cross-reactive anti-MOG idiotype may represent an important target for the anti-idiotypic regulation of demyelinating antibody responses in CREAE.

Protein kinase C activity in soluble fractions from glial cells in primary culture and subcultures

Protein kinase C (calcium + phospholipid-dependent kinase) activity has been measured in soluble 100,000 g fractions from mixed glial cells in primary culture; in 12 day cultures the specific activity (mean +/- S.D.) was 184 +/- 10 pmol 32P incorporated/10 min/mg protein. In glial cell subcultures lacking protoplasmic astrocytes protein kinase C specific activity was lower. An inhibitor of protein kinase C in 100,000 g supernatants was removed by chromatography through DE-52 anion exchange resin increasing the specific activity of the calcium + phospholipid-dependent kinase about 20 times. Protein kinase C was also associated with membrane fractions from glial cells; the membrane-associated enzyme had a higher specific activity than in the cytoplasm.

Lymphocytic activation in peripheral blood and cerebrospinal fluid during the course of chronic relapsing experimental allergic encephalomyelitis

A monoclonal antibody against the human interleukin-2 receptor (anti-Tac) has been found to cross-react with an antigen on the surface of guinea pig leucocytes. Cells marking with anti-Tac and with an anti-pan T cell monoclonal antibody have been quantitated in the peripheral blood and cerebrospinal fluid (CSF) of guinea pigs with chronic relapsing experimental allergic encephalomyelitis (CR-EAE). T cells account for about 90% of peripheral blood leucocytes in all animals whilst in the CSF, T cells are the major contributor only when there is a pleocytosis. The proportion of T cells marking with anti-Tac, a measure of T cell activation, in blood and CSF of control animals is 12%, rising to 23% in blood in the post-acute phase of the disease. However, a fall in the blood Tac/T ratio to 13% occurs during the first 10 days of relapse with a subsequent rise to 30-35%. This change is related to the time after onset of relapse irrespective of the subsequent course of the disease. From first relapse onwards CSF lymphocytes show a greater level of activation than lymphocytes from paired peripheral blood samples but the proportion of Tac+ cells in CSF does not increase with increasing CSF pleocytosis. The data is consistent with migration of activated T cells from blood to CSF at the onset of relapse.

Infection of rat brain primary cell cultures with an avirulent A7 strain of semliki forest virus

The ability of A7 Semliki Forest Virus (SFV) to infect primary brain cell cultures has been examined using cultures prepared from 1-2-day neonatal rat cerebral hemispheres. These cultures, characterised immunocytochemically using cell-specified markers, contain mainly GFAP+ protoplasmic astrocytes and smaller multiprocessed A2B5+ cells, probably fibrous astrocytes. 10% of the cells are GC+ oligodendrocytes and some neurones are also present. These cultures support virus growth and a cytopathic effect was observed. Using double labelling techniques with the cell-specific markers and anti-SFV antibody A7 has been shown to readily infect cells which carry either the A2B5+ antigen or galactocerebroside marker. Protoplasmic astrocytes (GFAP+/A2B5-) are not readily infected under the conditions used. The protein labelling studies using [35S]methionine show that host cell protein synthesis is not completely shut off and continues in the astrocyte protein region. These results suggest that cells derived from a common A2B5+, GFAP-, GC- progenitor glial cell, i.e. GC+ oligodendrocytes and A2B5+/GFAP+ fibrous astrocytes, are more readily infected than other brain cell types including the protoplasmic astrocytes.

Quantitative analysis of the cellular constituents of the cerebrospinal fluid in chronic relapsin experimental allergic encephalomyelitis

Cerebrospinal fluid (CSF) was taken from guinea pigs in various stages of chronic relapsing experimental allergic encephalomyelitis (CR-EAE). The leucocytes in CSF samples were counted and subjected to immunocytochemical analysis using monoclonal antibodies selectively recognising guinea pig T cells, macrophages or Ia antigens. The results showed that total leucocyte numbers and the proportion of macrophages in CSF were elevated in the acute phase of CR-EAE but samples of CSF from animals in early relapse did not show a significant elevation in leucocyte count or macrophage content. In addition the level of T cell activation was higher in CSF than in peripheral blood during disease and was highest in the acute and immediately post-acute phases of the CR-EAE.

Stimulation of protein phosphorylation in mixed glial cell primary cultures and subcultures by the phorbol ester 12-o-tetradecanoylphorbol-13-acetate (TPA)

Glial cell primary cultures consisting of protoplasmic and fibrous astrocytes, oligodendrocytes and progenitor glial cells incubated in medium containing 0.5% foetal calf serum and treated with 25 nM 12-o-tetradecanoylphorbol-13-acetate (TPA) for periods between 15 and 60 min showed a stimulation of protein phosphorylation which was most prominent in a polypeptide with a molecular weight of about 80,000 Da. Glial subcultures consisting mainly of Type 2 astrocytes, oligodendrocytes and progenitor glia showed a similar TPA stimulation of 80,000 Da protein phosphorylation detectable within 1 min of phorbol ester addition. TPA treatment of primary glial cultures led to an enhancement of phospholipid turnover but exposure of primary glial cultures to concentrations of TPA up to 250 nM caused no morphological change in protoplasmic astrocytes. 4-Phorbol (4-PH) or dimethylsulfoxide (DMSO) was without effect on protein phosphorylation or lipid turnover in glial cultures.

Activated T-cells and macrophages in the cerebrospinal fluid and the spinal meningeal exudate in chronic relapsing experimental allergic encephalomyelitis

An analysis has been made of the cell types which mark with monoclonal antibodies against T cells, macrophages and the IL-2 receptor (anti-Tac) in the blood, cerebrospinal fluid (CSF) and spinal meningeal exudates taken from guinea pigs in the relapse and remission stages of chronic relapsing experimental allergic encephalomyelitis (CR-EAE). Whilst the T-cell and macrophage content of blood remained unchanged throughout the course of CR-EAE, T cells accounted for the majority of the CSF pleocytosis associated with relapsing disease but both T cells and macrophages populated the meningeal exudate in substantial numbers. Activated T cells (Tac+) rose in number in blood only after the onset of relapse but formed a far higher proportion of the CSF pleocytosis or meningeal exudate than in paired blood samples. Meningeal exudate cells from Freunds adjuvant-inoculated, but not uninoculated animals, also showed an increase in Tac+ cell levels. In addition, the meningeal exudate contained a substantial number of cells which did not label with anti-T or anti-macrophage antibodies and which did not vary in absolute numbers throughout the course of disease.

Recovery and characterisation of immune cells from the central nervous system in chronic relapsing experimental allergic encephalomyelitis (CREAE)

I~:une cells have been isolated frc~l the spinal cord parenchyma and meninges of strain 13 guinea pigs with CREAE. At various stage of disease animals were extensively perfused, the cord stripped of meninges and the parenchyma chopped and sieved. Percoll gradient separation of cell suspensions allowed the recovery of 2xi06 cells/cord (and 2xi05 cells froa: the stripped meninges) in samples from relapsing animals. Even from uninoculated anin~is or anin~is inoculated with CFA only about 3xi0 5 cells could be recovered from cord preparations. In~L:unocytochemical analysis revealed that cel is from control animals were 80% macrophage/monocytes whilst cells from relapsing an~.Tals averaged 70% T cells. Functional studies using Terasaki assays showed that the inm~une cells recovered from cord parenchy~m responded to PtLA and PPD but only samples fran relapse and chronic animals proliferated in response to ~L~P. Analysis of the results from paired peripheral blood monocytes (PB~:) and parenchyn~l inflammatory cel I preparations indicated that 2/7 PBM samples and 5/7 parenchymal samples from relapse animals responded to MDP campared with 0/4 PBM and parenchymal samples from remission animals. An analysis of the phenotypic and functional properties of cells recovered from meningeal, parenchynel and peripheral blood cenlparhr, ents at various stages of CREAE will be presentecL