B. Höjeberg

Cytokine production in the central nervous system of Lewis rats with experimental autoimmune encephalomyelitis: dynamics of mRNA expression for interleukin-10, interleukin-12, cytolysin, tumor necrosis factor α and tumor necrosis factor β

The kinetics of mRNA expression in the central nervous system (CNS) for a series of putatively disease-promoting and disease-limiting cytokines during the course of experimental autoimmune encephalomyelitis (EAE) in Lewis rats were studied. Cytokine mRNA-expressing cells were detected in cryosections of spinal cords using in situ hybridization technique with synthetic oligonucleotide probes. Three stages of cytokine mRNA expression could be distinguished: (i) interleukin (IL)-12, tumor necrosis factor (TNF)-beta (= lymphotoxin-alpha) and cytolysin appeared early and before onset of clinical signs of EAE; (ii) TNF-alpha peaked at height of clinical signs of EAE; (iii) IL-10 appeared increasingly at and after clinical recovery. The early expression of IL-12 prior to the expression of interferon-gamma (IFN-gamma) mRNA shown previously is consistent with a role of IL-12 in promoting proliferation and activation of T helper 1 (Th1) type cells producing IFN-gamma. The TNF-beta mRNA expression prior to onset of clinical signs favours a role for this cytokine in disease initiation. A pathogenic effector role of TNF-alpha was suggested from these observations that TNF-alpha mRNA expression roughly paralleled the clinical signs of EAE. This may be the case also for cytolysin. IL-10-expressing cells gradually increased to high levels in the recovery phase of EAE, consistent with a function in down-regulating the CNS inflammation. From these data we conclude that there is an ordered appearance of putative disease-promoting and -limiting cytokines in the CNS during acute monophasic EAE.

CD8 is critically involved in lymphocyte activation by a T. brucei brucei-released molecule

T. brucei brucei released a lymphocyte triggering factor (TLTF), which triggered purified CD8+, but not CD4+, T cells to interferon gamma (IFN-gamma) mRNA expression and secretion and to [3H]thymidine incorporation. TLTF also induced mRNA for transforming growth factor beta, but not for interleukin-4. The action of this TLTF on mononuclear cell (MNC) cultures was blocked by anti-CD8 antibodies and by soluble CD8. MNCs from a mutant mouse strain lacking CD8 expression were not triggered by TLTF. IFN-gamma provides a growth stimulus for T. brucei brucei, and infected CD8- mice had much lower parasitemia and survived longer than CD8+ mice. The host-parasite interaction in experimental African trypanosomiasis thus involves parasite release of TLTF, which by binding to CD8 triggers CD8+ cells to produce the parasite growth-promoting cytokine IFN-gamma.

Cytokines in relapsing experimental autoimmune encephalomyelitis in DA rats: persistent mRNA expression of proinflammatory cytokines and absent expression of interleukin-10 and transforming growth factor-β

Experimental autoimmune encephalomyelitis (EAE) in rats is typically a brief and monophasic disease with sparse demyelination. However, inbred DA rats develop a demyelinating, prolonged and relapsing encephalomyelitis after immunization with rat spinal cord in incomplete Freunds adjuvant. This model enables studies of mechanisms related to chronicity and demyelination, two hallmarks of multiple sclerosis (MS). Here we have investigated, in situ, the dynamics of cytokine mRNA expression in the central nervous system (CNS) and peripheral lymphoid organs (lymph node cells and splenocytes) of diseased DA rats. We demonstrate that peripheral lymphoid cells stimulated in vitro with encephalitogenic peptides 69-87 and 87-101 of myelin basic protein responded with high mRNA expression for proinflammatory cytokines; interferon-gamma, interleukin-12 (IL-12), tumour necrosis factors alpha and beta, IL-1 beta and cytolysin. A high expression of mRNA for these proinflammatory cytokines was also observed in the CNS where it was accompanied by classical signs of inflammation such as expression of major histocompatibility complex class I and II, CD4, CD8 and IL-2 receptor. The expression of mRNA for proinflammatory cytokines was remarkably long-lasting in DA rats as compared to LEW rats which display a brief and monophasic EAE. Furthermore, mRNAs for putative immunodownmodulatory cytokines, i.e. transforming growth factor-beta (TGF-beta), IL-10 and IL-4 were almost absent in DA rats, in both the CNS and in vitro stimulated peripheral lymphoid cells, while their levels were elevated in the CNS of LEW rats during the recovery phase. We conclude that the MS-like prolonged and relapsing EAE in DA rats is associated with a prolonged production of proinflammatory cytokines and/or low or absent production of immunodownmodulatory cytokines.

Myelin antigen reactive T cells in cerebrovascular diseases

T cell reactivities to the putative autoantigens myelin basic protein (MBP), MBP pcptides with amino acid residues 110–128 and 148–165, and myelin proteolipid protein (PLP) were examined in patients with acute ischaemic cerebrovascular disease (CVD) and, for comparison, in patients with inflammatory neurological diseases and other neurological diseases. A quantitative measure of these T cell reactivities was obtained by assessing numbers of T cells among blood and cerebrospinal fluid (CSF) mononuclear cells that secreted IFN‐γ in response to antigen in vitro. Higher numbers of T cells reactive with each of these four antigens were detected in peripheral blood from patients with CVD compared with patients of the two control groups. Among blood cells from the CVD patients, their average number was 2·3–4·2/105 mononuclear cells. MBP reactive T cells were several‐fold enriched in the CSF of CVD patients. The findings strongly suggest that brain damage in context with acute CVD leads to an in vivo expansion of myelin reactive T cells.

Facial nerve transection causes expansion of myelin autoreactive T cells in regional lymph nodes and T cell homing to the facial nucleus

Nervous tissue expression of immunological signal and recognition molecules, as well as lymphoid tissue immune responses after facial nerve trauma was studied in male rats of the Lewis and Brown Norway (BN) strains. In both rat strains nerve transection caused within four days the appearance of IFN-gamma-like immunoreactivity in the cytoplasm of axotomized motor neurons and an induction of MHC class I and II, and CD4 molecules on surrounding glial cells to a similar extent. T lymphocytes also infiltrated the facial nuclei ipsilateral to the axotomy in all animals. The number of autoreactive T cells in superficial cervical lymph nodes, which in response to whole myelin or peptides of myelin basic protein (MBP) secreted IFN-gamma increased markedly after axotomy. This response was more conspicuous in Lewis rats, which are susceptible to experimental allergic encephalomyelitis (EAE), than in BN rats, which are EAE resistant. A proportion of the axotomized Lewis rats also developed widespread perivascular infiltration of mononuclear cells in the CNS, reminiscent of EAE. Hypothetically, a strong expansion of myelin autoreactive IFN-gamma producing T cells secondary to nerve trauma may have immunopathological consequences in genetically predisposed individuals. It is also possible that myelin reactive T cells, whether recruited to the lesioned nerve, could have impact on macrophage function during Wallerian degeneration in the distal stump.

The major histocompatibility complex influences myelin basic protein 63‐88‐induced T cell cytokine profile and experimental autoimmune encephalomyelitis

Polymorphism of the major histocompatibility complex (MHC) influences susceptibility to experimental autoimmune encephalomyelitis (EAE) induced by myelin basic protein (MBP) in rats. Current concepts relate such influences to the capacity of class II molecules to present relevant peptides to autoreactive T cells. We have here analyzed the MHC influence on the immune response and the development of EAE after immunization with the immunodominant peptide MBP‐63–88. Analysis of MHC‐congenic LEWIS strains showed that RT1a, RT1c and RT1l haplotypes are permissive for disease induction, whereas RT1d and RT1u are resistant. All EAE responding strains showed peptide‐specific proliferation and interferon (IFN)‐γ secretion, but no early significant tendency to express interleukin (IL‐4) or transforming growth factor (TGF)‐β mRNA in lymphocytes in response to the MBP 63–88, 7 days post immunization (p.i.). Later, 14 days p.i., peptide‐specific induction of IL‐4 and TGF‐β occurred in RT1l rats. Among the EAE non‐responders strains, only the RT1u rats showed an immune response to MBP 63‐88. This response, however, was qualitatively different from the immune response in the EAE‐susceptible strains. Thus, there was no proliferation and only moderate IFN‐γ production in response to peptide, but in contrast, a significant and early peptide‐induced IL‐4 and TGF‐β response was observed. The data suggest that the MHC‐associated susceptibility to EAE is partly related to the ability to mount a TH1‐like immune response while the MHC‐associated EAE resistance may either be related to MBP peptide non‐responsiveness or to peptide recognition and induction of a qualitatively different and disease down‐regulatory immune response.

A Trypanosoma brucei brucei-Derived Factor that Triggers CD8+ Lymphocytes to Interferon-γ Secretion: Purification, Characterization and Protective Effects In Vivo by Treatment with a Monoclonal Antibody against the Factor

A protein factor that stimulates CD8+ lymphocytes to produce and secrete IFN‐γ has been purified from Trypanosoma brucei brucei (T.b. brucei). This was accomplished by raising monoclonal antibodies (MoAbs) against a fraction of T.h. brucei obtained by gel filtration, whieh contained high levels of material inducing rat mononuelear cells (MNC) to IEN‐y production. MoAbs from four hybridomas strongly inhibited trypanosome‐induced IFN‐γ production. One of them (MO1) was used for purification of the trypanosome‐derived lymphocyte triggering factor (TLTF) by affinity chromatography, SDS electrophoresis of the purified TLTF displayed a band of 42‐45 kDa MW. Gel filtration of homogenates of whole parasites yielded several peaks of IFN‐γ‐inducing activity with a lowest MW of 41 46 kDa. Bioactivity of all peaks was blocked by MO1. suggesting that a single molecule, or a single epitope of additional molecules, is responsible for the different peaks with IFN‐γ‐indueing activity. IFN‐γ released from MNC stimulates T.b. brucei growth. Blocking of TLTF in vitro with MO1 inhibited MNC‐supported growth of the parasites. To study the in vivo relevance of TLTF in the course of experimental African trypanosomiasis, MO1 was used to treat rats and mice at different times after infection. Treatments instituted at different time‐points after infection suppressed parasite growth, abrogated the IFN‐γ production by splenocytes indueed by the infection and prolonged survival of the animals. The data support the hypothesis that TLTF and IFN‐γ have a erueial regulatory function in the parasite host interactions and that these moleeules influence the disease eourse during experimental African trypanosomiasis.

Multiple Sclerosis: Cells Secreting Antibodies Against Myelin-Associated Glycoprotein are Present in Cerebrospinal Fluid

We evaluated the B‐cell response in cerebrospinal fluid (CSF) and blood by enumerating cells secreting antibodies to myelin‐associated glycoprotein (MAG) and, for reference, to myelin basic protein (MBP), two myelin components which may constitute targets for autoimmune attack in multiple sclerosis (MS). Among 25 untreated MS patients, 12 had cells in CSF secreting anti‐MAG IgG antibodies (mean value 1 per 1429 CSF cells) and three also had cells secreting anti‐MAG antibodies of the IgM Isotype but at lower levels. In CSF from 2 out of 10 MS patients examined, anti‐MAG and anti‐MBP IgG antibody‐secreting cells were present concurrently. Antibody‐secreting cells were less frequent in blood and bone marrow, reflecting compartmentalization to CSF. Anti‐MAG antibody‐secreting cells were found in CSF from only I out of 27 control patients. The intrathecal production of anti‐MAG and anti‐MBP antibodies may be important in the pathogenesis of MS.

T Cells Recognizing Multiple Peptides of Myelin Basic Protein are Found in Blood and Enriched in Cerebrospinal Fluid in Optic Neuritis and Multiple Sclerosis

The cause of multiple sclerosis (MS) is unknown. Recently reported abnormal T‐cell responses to several myelin proteins and myelin basic protein (MBP) peptides in peripheral blood constitute one line of evidence that autoimmune mechanisms could be involved in the pathogenesis of the disease. Monosymptomatic unilateral optic neuritis (ON) is a common first manifestation of MS and important to examine for a possible restriction of the T‐cell repertoire early in the disease. T‐cell activities to MBP and the MBP amino acid sequences 63–88, 110–128 and 148–165 were examined by short‐term cultures of mononuclear cells from cerebrospinal fluid (CSF) and blood in the presence of these antigens, and subsequent detection and counting of antigen‐specific T cells that responded by interferon‐gamma (IFN‐γ) secretion. Most patients with MS and ON had MBP and MBP peptide‐reactive T cells in CSF, amounting to mean values of between about 1 per 2000 and 1 per 7000 CSF cells and without immunodominance for any of the peptides. Numbers were 10‐fold to 100‐fold lower in the patients′ blood. Values were similar in ON and MS, and no evidence was obtained for a more restricted T‐cell repertoire in ON. The MBP peptide‐recognizing T‐cell repertoire was different in CSF than in blood in individual patients with ON and MS, thereby giving further evidence for an autonomy of the autoimmune T‐cell response in the CSF compartment. No relations were observed between numbers of autoreactive T cells and presence of oligoclonal IgG bands in CSF or abnormalities on magnetic resonance imaging of the brain in ON or clinical variables of MS. The high numbers of MBP and MBP peptide‐reactive T cells could play a role in the pathogenesis of ON via secretion of effector molecules, one of them being IFN‐γ, as well as in the transfer of ON to MS.

Autoreactive T and B cell responses to myelin antigens after diagnostic sural nerve biopsy

To study whether nervous tissue trauma provokes myelin antigen autoreactive T and B cell responses in humans we examined consecutive blood samples from 7 patients with polyneuropathy undergoing diagnostic sural nerve biopsy and 8 control patients undergoing other types of minor surgery. The antigen-specific T cells were assessed by enumerating cells secreting interferon-gamma (IFN-gamma) in response to the myelin components P0, P2, myelin basic protein (MBP) and myelin associated glycoprotein (MAG), and to 4 selected MBP peptides. B cell mediated immunity was assessed by counting numbers of cells secreting antibodies directed against the myelin proteins. On day 7 after biopsy, there were 3-10-fold increased numbers of T and B cells reactive with P0, P2, MBP and MAG in blood of polyneuropathy patients compared to controls, while levels of cells recognizing purified protein derivate or responding to phytohemagglutinin (PHA) did not differ significantly. Comparison of prebiopsy levels on day 0 with post-biopsy levels on day 7 in the polyneuropathy patients revealed a significant increase in T cells recognizing P0, P2 and MAG, and in B cells secreting IgG antibodies against P0 and P2. On day 14 after nerve biopsy these differences were no longer seen. We suggest that in patients with polyneuropathy, sural nerve biopsy with the ensuing wallerian degeneration and myelin breakdown causes transiently increased levels of circulating myelin autoreactive T and B cells. It remains to be determined if this has a physiological role in nerve trauma responses and/or affects the clinicopathological course of the peripheral neuropathy.