DNA Changes in Spinal Cords of Rats with Experimental Allergic Encephalomyelitis

DNA levels were measured in the spinal cords of Lewis rats during the development of and recovery from experimental allergic encephalomyelitis (EAE). Spinal cord DNA was first increased 11 days after immunizing the rats with guinea pig myelin and rose to levels four times that of the Freund's adjuvant controls at day 14, then subsided after day 22. Spinal cord DNA was still 150% of control levels 60 days after immunization. These DNA changes were compared with fluctuations in spinal cord acid proteinase in the same animals. Acid proteinase activity in EAE spinal cord increased later than the rise in DNA and attained a level of 170% of control at days 15-17, then subsided. Spinal cord DNA was higher in rats immunized with whole myelin than in those administered equivalent amounts of purified myelin basic protein. Furthermore DNA was higher in spinal cords of rats immunized with a larger dose of myelin (1.0 mg) than with a lower amount (0.5 mg). Various protease inhibitors including pepstatin, nitrophenyl p-guanidino benzoate, polylysine, and dipropionyl rhein, previously shown to protect Lewis rats against EAE, suppressed the increase of DNA in the spinal cord. Measurement of DNA increases in the spinal cord of EAE animals provides a convenient reproducible measurement of the severity of inflammation in the CNS and provides an objective criterion for assessment of the efficacy of various agents screened as possible therapeutic treatment for multiple sclerosis.     

METABOLIC ACTIVITY OF CNS PROTEINS IN RATS AND MONKEYS WITH EXPERIMENTAL ALLERGIC ENCEPHALOMYELITIS (EAE)

—The metabolic activity of proteins from myelin and non-myelin fractions of slices of lesions in monkey brains and in spinal cords of Lewis rats with acute experimental allergic encephalomyelitis was investigated using [1-¹⁴C]leucine as a protein precursor. The uptake in vitro of [1-¹⁴C]leucine into the monkey EAE lesions was greatly increased in both the myelin and non-myelin fractions. Similar findings were made in spinal cord slices of the EAE rat with an average specific activity 341 per cent of control measured in proteins of purified myelin and 415 per cent of control in the non-myelin protein. The increased uptake appeared with the onset of paralytic symptoms 10–14 days after injection. The increased uptake did not appear to be a result of an increased amino acid pool size as measured with uniformly labelled l -leucine, valine, arginine and phenylalanine. The increase in specific activity of the myelin protein of the EAE rats was shown to be associated with the peaks characteristic of myelin protein when separated on polyacrylamide gels and the serial slices counted. Most of the radioactivity of both the control and EAE myelin protein migrated with the high molecular weight fraction, and the largest increase in radioactivity in myelin protein appeared in this fraction. Some increase in specific activity was also found in the basic and proteolipid proteins. Four different guinea-pig antigens were used to induce EAE: whole spinal cord, purified basic protein, purified myelin and basic protein + cerebroside. All caused paralytic symptoms and greatly increased incorporation in vitro of [1-¹⁴C]leucine into spinal cord proteins. The incorporation of [1-¹⁴C]leucine into slices of the inguinal and popliteal lymph nodes of the EAE and Freund's adjuvant control rats were measured and compared with the incorporation into the spinal cord non-myelin fractions. The specific activity of lymph node proteins was of the order of 10 × that of the non-myelin protein of the control spinal cord. Invasion of a moderate number of cells of the order of activity of these lymph nodes could account for the large increase in rate of protein synthesis in the EAE nervous tissue. It is concluded that much of the increased protein synthesis could be due to the inflammatory cells, although a small amount of the total increase appears to be associated with myelin protein. Other changes in metabolism of the CNS tissue of the EAE rat include a lower rate of lipid synthesis and a decreased activity of the tricarboxylic acid cycle.     

Suppression of experimental allergic encephalomyelitis in rats by mercuric chloride

Brown-Norway (BN) rats are uniquely susceptible to development of autoimmune phenomena and enlargement of lymph nodes and spleen after repeated injections of mercuric chloride. Despite its ability to produce autoimmunity, HgCl2 inhibited the development in BN rats of experimental allergic encephalomyelitis (EAE), another autoimmune process. The inhibition by mercury was probably due to lack of the normal absorption and granulomatous reaction to the EAE inoculum in the enlarged lymph nodes draining the inoculation site. Lewis rats did not develop enlarged nodes from HgCl2 treatment. Lewis lymph nodes absorbed the EAE inoculum abundantly and developed an extensive granulomatous reaction despite the mercury treatment, and there was only a slight inhibition of EAE. Therefore, the ability of HgCl2 to produce lymphadenopathy in BN rats may be responsible for the inability of these rats to absorb the inoculated antigen. The mercury-induced failure of absorption was manifested as an inhibition of EAE in BN rats.     

Neutral protease activity in lymphocytes of lewis rats with acute experimental allergic encephalomyelitis

Lymphocytes from popliteal and inguinal lymph nodes of Lewis rats with acute EAE as a result of injection of lyophilized guinea pig myelin in Freund's complete adjuvant exerted strong proteolytic activity at neutral pH toward myelin basic protein. After injection of myelin the level of proteolytic activity remained about the same as that in lymphocytes from Freund's adjuvant-injected controls until about day 10 after injection, just before the onset of paralytic symptoms; then the proteolytic activity increased to approximately double its former level. Myelin basic protein was hydrolyzed by whole lymphocytes, but more activity was unmasked by homogenization. Similar results were also obtained using lymphocytes from thymus of EAE and control animals. Lymphocytes with high levels of proteolytic activity were not absorbed by glass wool, did not stain with neutral red, nor did they phagocytose antibody-coated sheep red blood cells. Thymus and lymph node lymphocytes cleaved myelin basic protein to three major peptides and a fourth minor peptide, while spleen lymphocytes hydrolyzed basic protein at only one point resulting in two peptides whose molecular weights added up to that of myelin basic protein. The protease activity was inhibited by 5×10^–3 Mp-chloromercuribenzoate and by phenylmethyl sulfonyl fluoride, TPCK, and soybean trypsin inhibitor, therefore the enzymatic activity probably depends on a serine residue and a sulfhydryl group. The bulk of the enzymatic activity is mostly membrane bound with the highest specific activity and total activity contained in a lysosomal-mitochondrial fraction. In view of the infiltration of lymphocytes into the brain substance in acute EAE, it is suggested that these cells may contribute to the destruction of myelin which is usually attributed to the monocyte or macrophage.     

Brain and Spinal Cord Levels of Histamine in Lewis Rats with Acute Experimental Autoimmune Encephalomyelitis

Acute experimental autoimmune encephalomyelitis (EAE) was induced in Lewis rats by inoculation with guinea pig spinal cord homogenate emulsified with Mycobacterium tuberculosis-enriched complete Freund's adjuvant (CFA). Control rats were inoculated with CFA alone. Control and EAE rats were killed on days 7, 9, 11, and 13 postinoculation, and regional brain and spinal cord levels of histamine were determined. No regional differences in histamine content between control and EAE rats were seen on day 7 or 9 postinoculation. However, depending on the region, EAE rats exhibited significantly higher levels of histamine in their CNS on day 11 or 13 postinoculation or on both. Thus, regionally and temporally specific increases in brain and spinal cord levels of histamine develop concomitant with or just after the appearance (on day 10 postinoculation) of clinical signs of acute EAE, a finding suggesting that histamine may be involved in the development or expression of acute EAE in Lewis rats.     

Acid proteinases and other acid hydrolases in experimental allergic encephalomyelitis: pinpointing the source.

—In Lewis rats with experimental allergic encephalomyelitis (EAE), quantitative histochemical techniques were used to measure the activities of several hydrolytic enzymes, especially acid proteinases, and their levels were correlated with the concentrations of DNA and total lipids. Only those samples which contained lesions (visualized directly or in stained nearby sections) had elevated acid hydrolase activities. A lipase-esterase hydrolysing a fluorogenic substrate at pH 5.0 was more active in lesions; activity on the same substrate at pH 7.2 was not elevated. β-Glucuronidase rather than acid proteinase or cathepsin D was the enzyme showing the greatest differences. In a monkey with EAE, studied for comparison, a massive inflammatory lesion appeared to leak β-glucuronidase to a distance of about 2.5 mm beyond its margin.     

Autoimmune effector cells. III. Role of adjuvant and accessory cells in the in vitro induction of autoimmune encephalomyelitis

The present investigation shows that autoreactive effector cells that transfer experimental allergic encephalomyelitis (EAE) can be activated from spleens and lymph nodes of Lewis rats given a single injection of 25 micrograms myelin basic protein (BP) in incomplete Freund's adjuvant (IFA), despite the fact that the cell donors do not develop EAE. Rather, these donor rats are unresponsive to EAE when given an encephalitogenic emulsion of BP in complete Freund's adjuvant (CFA). Lymphoid cells from rats given a single injection of BP-IFA were almost as effective as cells from BP-CFA-treated rats with respect to transferring EAE after in vitro activation with BP or concanavalin A (Con A). Irrespective of whether donors received BP in IFA or CFA, BP-cultured spleen and lymph node cells (SpC and LNC, respectively) transferred EAE, whereas Con A-cultured SpC but not LNC exhibited effector cell activity. Con A-cultured LNC were able to transfer EAE if the cultures were reconstituted with irradiated adherent phagocytic cells (which could be obtained from normal Lewis rat spleens) or with conditioned medium from these adherent SpC. These findings indicate that accessory cells are required for in vitro induction of this T cell-mediated autoimmune response.     

Experimental allergic encephalomyelitis in male Lewis rats subjected to calorie restriction

This work analyzes the effect of calorie restriction on the development of experimental allergic encephalomyelitis (EAE) in Lewis rats. Plasma levels of ACTH, corticosterone, prolactin and growth hormone (GH) and mitogenic responses in submaxillary lymph nodes were measured. Male Lewis rats (6 weeks-old) were submitted to a calorie restriction equivalent to 66% of food restriction or to a normal diet. Fifteen days later, rats were injected with complete Freund's adjuvant plus spinal chord homogenate (SCH) or with complete Freund's adjuvant alone. Rats were monitored daily for clinical signs of EAE and were killed on day 15 after immunization. Only rats subjected to normal diet exhibited clinical signs of the disease. The increase in plasma ACTH and corticosterone found after SCH immunization in controls was not detectable in calorie restricted rats. Likewise, the correlation between circulating ACTH and corticosterone was no longer found after calorie restriction. Generally, calorie restriction by itself augmented plasma ACTH or corticosterone and this increase was not further amplified by SCH immunization. Only calorie restricted rats exhibited augmented plasma prolactin levels after SCH immunization, and decreased plasma GH levels regardless of immunization. Calorie restriction depressed the mitogenic response of lymphoid cells to concanavalin A but not to lipopolysaccharide. Calorie restricted rats did not exhibit augmented mitogenic response to concanavalin A following SCH immunization as those found in controls. The results are compatible with the view that the course of EAE can be significantly modified by caloric restriction.     

切除肾上腺对大鼠自身免疫性脑脊髓炎敏感性的影响

目的研究肾上腺切除导致下丘脑-垂体-肾上腺(HPA)轴功能缺陷对Wistar大鼠诱发的实验性自身免疫性脑脊髓炎(EAE)的敏感性的影响。方法 Wistar大鼠单、双侧肾上腺切除后,用GPSCH-CFA诱导各组大鼠产生EAE,并观察临床评分、MBP抗体、皮质醇、Th1细胞因子和脑组织Bcl-2/Bax蛋白表达的变化,并与Lewis大鼠的EAE模型进行比较。结果 GPSCH-CFA诱导后,双侧切除肾上腺的Wistar大鼠平均神经症状评分最高为4.60分,发病率为100%,血清中皮质醇水平明显降低和MBP抗体水平明显升高,IFN-γ、TNF-α和IL-2水平均明显升高,大脑皮层和下丘脑中Bcl-2蛋白表达均明显降低和Bax蛋白表达均明显升高。结论双侧肾上腺切除导致HPA功能缺陷能显著增加Wistar大鼠诱发EAE的敏感性。     

Experimental allergic encephalomyelitis: clinical disease and enhanced cellular transfer in the absence of lymphocyte proliferative responses against syngeneic MBP

Experimental allergic encephalomyelitis (EAE) was induced in Lewis rats using several different immunization protocols, and draining lymph node cells from these animals were assayed for proliferation against heterologous, homologous, and syngeneic MBP, and syngeneic spinal cord. Proliferative responses were largely stimulated by nonsyngeneic antigenic determinants and correlated better with the antigen used to induce EAE than with signs of autoimmune disease. Lymph node cells from rats immunized with either guinea pig spinal cord or syngeneic MBP did not proliferate measurably when restimulated in vitro with syngeneic MBP, yet lymphoid cells from these animals were enhanced in their capacity to transfer EAE following in vitro stimulation with syngeneic MBP.     

Chronic permeability of the central nervous system to mononuclear cells in experimental allergic encephalomyelitis in the Lewis rat.

In order to assess whether experimental allergic encephalomyelitis (EAE), a putative animal model for multiple sclerosis (MS), is an ongoing chronic disorder, we have studied the permeability of spinal cords of Lewis rats with EAE to 3H-uridine- or 3H-thymidine-labeled lymphoid cells obtained from thymuses of naive donors or from draining lymph nodes of donors injected with guinea pig spinal cord + complete Fruend's adjuvant (CFA), guinea pig myelin basic protein + CFA, or with CFA alone. During the acute clinical phase of EAE there is a high-level infiltration of 3H-thymidine- or 3H-uridine-labeled cells into the spinal cords. After clinical recovery from EAE up to 58 days post-inoculation, there is a low-level infiltration of 3H-thymidine-labeled cells into the spinal cords. A similar infiltration into the spinal cords by 3H-uridine-labeled cells was not detected. Donor cells from animals immunized with CFA alone showed similar levels of infiltration into the spinal cords of animals with EAE as donor cells from animals immunized with the encephalitogenic emulsion. Spinal cords from recipients immunized with CFA alone showed no increased permeability to labeled cells. Heat-killed labeled cells did not migrate into the spinal cords of animals with EAE. We conclude that a) EAE is a chronic disease and in this regard is a valid model for MS; and B) in the chronic phase of EAE, recently divided cells (3H-thymidine-labeled cells) show higher levels of migration into the target tissue than 3H-uridine-labeled cells.     

Limiting dilution analysis of the frequency of antigen-reactive lymphocytes isolated from the central nervous system of Lewis rats with experimental allergic encephalomyelitis

Lymphocytes were isolated from the spinal cord and draining lymph nodes of Lewis rats with acute experimental allergic encephalomyelitis (EAE) 12 days after immunization with myelin basic protein (MBP) and tetanus toxoid (TT). An average of 8.0 +/- 2.0 X 10(6) cells was obtained from the spinal cord. Of these 71.1 +/- 8.6% expressed the helper-T-cell marker W3/25 and 14.8 +/- 6.2% expressed the killer/suppressor-T-cell marker OX8. By limiting dilution analysis of cells exhibiting an antigen-specific proliferative response, the average frequencies of cells reactive to MBP and TT were 3.36 +/- 2.4 and 7.60 +/- 4.1 per 10(4), respectively. In the draining lymph nodes, the frequencies of cells reactive to MBP and TT were 2.24 +/- 1.7 and 2.69 +/- 2.5 per 10(4). At a relatively early stage of clinical EAE, MBP-reactive T cells comprise only a small minority of the cells which can be isolated from the spinal cord; lymphocytes reactive to a protein antigen irrelevant to EAE pathogenesis are present in comparable numbers. This finding suggests that most of these cells accumulate as a result of mechanisms not specific for MBP-reactive lymphocytes.     

Tracking of V beta 8.2-positive encephalitogenic T cells by complementarity-determining region 3 spectratyping and subsequent Southern blot hybridization in Lewis rats after neuroantigen sensitization

Pathogenic T cells in organ-specific autoimmune diseases use a limited number of TCR alpha- and beta-chains. In experimental autoimmune encephalomyelitis (EAE) induced in Lewis rats by immunization with myelin basic protein, encephalitogenic T cells mainly use Vbeta8.2 TCR and clonal expansion of the Vbeta8.2 spectratype containing the EAE-specific complementarity-determining region 3 (CDR3) sequence, DSSYEQYFGPG, is found in the spinal cord throughout the course of clinical EAE. In the present study we performed temporal and spatial analyses of Vbeta8.2 spectratype expansion by CDR3 spectratyping and subsequent DNA hybridization with a probe specific for the encephalitogenic CDR3 sequence to elucidate the kinetics of encephalitogenic T cells during the induction phase after neuroantigen sensitization. It was demonstrated that Vbeta8.2 spectratype expansion and/or the positive signal in Southern blot were first detected in the regional lymph nodes as early as day 3 postimmunization and was disseminated over the lymphoid organs by day 6. Because perfusion of immunized rats with PBS erased the positive signals on day 3 postimmunization, the majority of Vbeta8.2-positive encephalitogenic T cells at the very early stage would reside within the lymphatic or blood vessels. Furthermore, removal of the draining lymph node 1, 3, and 6 days after immunization in the foot pad did not ameliorate clinical EAE. These findings strongly suggest that encephalitogenic T cells disseminate throughout the whole body very rapidly after sensitization. Analysis of pathogenic T cells at the clonal level provides useful information for designing effective immunotherapy.     

ACETYLCHOLINE,CHOLINE AND CHOLINE ACETYLTRANSFERASE ACTIVITY IN THE DEVELOPING BRAIN OF NORMAL AND HYPOTHYROID RATS1

Abstract— Acetylcholine, choline and choline acetyltransferase activity were measured in the whole brains of normal and hypothyroid rats during development. At 1 day postpartum, brain acetylcholine was 73 per cent of adult levels. Propylthiouracil-induced hypothyroidism up to age 20 days did not alter brain acetylcholine concentrations, but at 30 days resulted in significantly decreased levels. At day 1, brain choline was 20 per cent higher than adult levels and decreased between days 8 and 10. In hypothyroid rats this phenomenon did not occur until days 15–20. At day 1 postnatally, choline acetyltransferase activity was only 7 per cent of adult levels, then between days 5 and 20 rose to 77 per cent of adult levels. Beginning at day 8, hypothyroidism resulted in significantly decreased enzyme levels. This effect could be reversed at day 17 by concurrent tri-iodothyronine substitution therapy. In hypothyroid rats, maximum brain choline acetyltransferase activity was 30 per cent less than normal adult levels.     

Endocrine and lymphoid interrelations in rats with hereditary arterial hypertension

Functional state of the adrenals and structural organization of the thymus and lymph nodes in the rats with hereditary stress-induced arterial hypertension (HSIAH) have been investigated in the control and against the background of a prolonged staying under conditions of a moderate cold. In comparison with the initial population of Wistar rats, the test animals demonstrate an elevated level of corticosterone, decreased mass and size of the thymus, the size of the popliteal lymph nodes is increased at the expense of the structural components of the medulla. In 7 weeks of living in cold secretion of adrenal steroids in Wistar rats increases greatly and it is noticeably less in the HSIAH rats. Structural changes in the thymus are also insignificant, but reaction of the lymph nodes is important. Their size sharply diminishes at the expense of certain structural components of the medulla. A shift towards mature forms of the plasmocytic line cells takes place.     

Cerebrospinal fluid dendritic cells infiltrate the brain parenchyma and target the cervical lymph nodes under neuroinflammatory conditions

Background

In many neuroinflammatory diseases, dendritic cells (DCs) accumulate in several compartments of the central nervous system (CNS), including the cerebrospinal fluid (CSF). Myeloid DCs invading the inflamed CNS are thus thought to play a major role in the initiation and perpetuation of CNS-targeted autoimmune responses. We previously reported that, in normal rats, DCs injected intra-CSF migrated outside the CNS and reached the B-cell zone of cervical lymph nodes. However, there is yet no information on the migratory behavior of CSF-circulating DCs under neuroinflammatory conditions.

Methodology/Principal Findings

To address this issue, we performed in vivo transfer experiments in rats suffering from experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis. EAE or control rats were injected intra-CSF with bone marrow-derived myeloid DCs labeled with the fluorescent marker carboxyfluorescein diacetate succinimidyl ester (CFSE). In parallel experiments, fluorescent microspheres were injected intra-CSF to EAE rats in order to track endogenous antigen-presenting cells (APCs). Animals were then sacrificed on day 1 or 8 post-injection and their brain and peripheral lymph nodes were assessed for the presence of microspheres⁺ APCs or CFSE⁺ DCs by immunohistology and/or FACS analysis. Data showed that in EAE rats, DCs injected intra-CSF substantially infiltrated several compartments of the inflamed CNS, including the periventricular demyelinating lesions. We also found that in EAE rats, as compared to controls, a larger number of intra-CSF injected DCs reached the cervical lymph nodes. This migratory behavior was accompanied by an accentuation of EAE clinical signs and an increased systemic antibody response against myelin oligodendrocyte glycoprotein, a major immunogenic myelin antigen.

Conclusions/Significance

Altogether, these results indicate that CSF-circulating DCs are able to both survey the inflamed brain and to reach the cervical lymph nodes. In EAE and maybe multiple sclerosis, CSF-circulating DCs may thus support the immune responses that develop within and outside the inflamed CNS.     

Expression of Ccl11 associates with immune response modulation and protection against neuroinflammation in rats

Multiple sclerosis (MS) is a polygenic disease characterized by inflammation and demyelination in the central nervous system (CNS), which can be modeled in experimental autoimmune encephalomyelitis (EAE). The Eae18b locus on rat chromosome 10 has previously been linked to regulation of beta-chemokine expression and severity of EAE. Moreover, the homologous chemokine cluster in humans showed evidence of association with susceptibility to MS. We here established a congenic rat strain with Eae18b locus containing a chemokine cluster (Ccl2, Ccl7, Ccl11, Ccl12 and Ccl1) from the EAE- resistant PVG rat strain on the susceptible DA background and utilized myelin oligodendrocyte glycoprotein (MOG)-induced EAE to characterize the mechanisms underlying the genetic regulation. Congenic rats developed a milder disease compared to the susceptible DA strain, and this was reflected in decreased demyelination and in reduced recruitment of inflammatory cells to the brain. The congenic strain also showed significantly increased Ccl11 mRNA expression in draining lymph nodes and spinal cord after EAE induction. In the lymph nodes, macrophages were the main producers of CCL11, whereas macrophages and lymphocytes expressed the main CCL11 receptor, namely CCR3. Accordingly, the congenic strain also showed significantly increased Ccr3 mRNA expression in lymph nodes. In the CNS, the main producers of CCL11 were neurons, whereas CCR3 was detected on neurons and CSF producing ependymal cells. This corresponded to increased levels of CCL11 protein in the cerebrospinal fluid of the congenic rats. Increased intrathecal production of CCL11 in congenic rats was accompanied by a tighter blood brain barrier, reflected by more occludin(+) blood vessels. In addition, the congenic strain showed a reduced antigen specific response and a predominant anti-inflammatory Th2 phenotype. These results indicate novel mechanisms in the genetic regulation of neuroinflammation.     

Antibody to myelin constituents: A possible factor in induction of cell-mediated demyelination

Results from this laboratory have demonstrated that¹⁴C-labeled myelin opsonized with antibodies raised to purified CNS myelin in rabbit is phagocytized by cultured macrophages in larger amounts than untreated myelin or myelin opsonized with preimmune serum. The cultured macrophages produced high amounts of radioactive cholesterol ester and triglyceride from the antibody-treated myelin while much less was formed from preimmune serum-treated or untreated myelin. Antiserum to galactocerebroside also greatly enhanced the formation of radioactive cholesterol ester, while that to myelin basic protein as well as to other myelin constituents had little or no effect. Serum from Lewis rats with acute EAE 13–14 days after immunization with whole CNS myelin also stimulated radioactive cholesterol ester formation compared to serum from Freund's adjuvant-injected controls (FAC). Serum from EAE rats as a result of myelin basic protein injection was as active as that from rats with whole myelin injection. No galactocerebroside antibody could be demonstrated in the EAE sera, although a strong immunostaining to myelin basic protein and proteolipid protein was demonstrated. IgG prepared from EAE serum also showed stimulatory effects compared to IgG from FAC serum, but much of the activity was lost, and the possibility that other factors may be involved is discussed. These experiments provide evidence that myelin phagocytosis and digestion by macrophages is enhanced by the presence of antibody to myelin. In EAE this antibody may leak into CNS with the breakdown of the blood-brain barrier. A humoral involvement in demyelination in EAE is implicated, and these findings may be extended eventually to the demyelinative mechanism in multiple sclerosis where IgG is found in large amounts in the CNS.Special Issue Dedicated to Dr. Abel Lajtha.     

Immunoregulation of experimental allergic encephalomyelitis: conditions for induction of suppressor cells and analysis of mechanism.

We determined requirements for the induction of immunoregulatory suppressor cells in experimental allergic encephalomyelitis (EAE) in Lewis rats. Pretreatment of rats with myelin basic protein (BP) in incomplete Freund's adjuvant (IFA) stimulates the proliferation of suppressor cells that localize in lymph nodes and spleen (but not thymus) and exert control over the development of clinical EAE. Dosage studies revealed that 3 X 10(7) suppressor cells can adoptively transfer suppression to syngeneic recipients. Transferred unresponsiveness wanes within 3 weeks, indicating that the suppressor cells are short-lived lymphocytes, although actively induced unresponsiveness persists for at least 8 weeks, probably as a result of continual proliferation under the influence of antigen. No evidence was obtained to suggest that antigen carry-over or blocking antibody production accounts for adoptive transfer of unresponsiveness. Suppressor cells apparently act at the inductive phase of the immune response since they had no inhibitory effect on adoptive transfer of disease by effector lymph node cells. Other mechanisms also may play a role in unresponsiveness to EAE, since rats pretreated i.v. with high dosages of soluble BP were temporarily rendered unresponsive, although suppressor cells could not be detected in these animals.     

Immunosuppression of experimental allergic encephalomyelitis. III. In vitro evidence for induction of suppressor T lymphocytes in draining lymph node cells of animals immunized with myelin basic protein complexed to lipopolysaccharides

We recently demonstrated that Lewis rats immunized with bacterial lipopolysaccharides (LPS) precomplexed to guinea pig myelin basic protein (BP) in complete Freund's adjuvant were less effective in inducing experimental allergic encephalomyelitis (EAE) than BP-immunized controls. When tested in vitro both lymph node cells (LNC) and spleen cells (SpC) of animals immunized with BP-LPS were less effective in proliferative responses to various mitogens, which included phytohemagglutinin, concanavalin A, purified protein derivative of tuberculin, LPS, and BP. Of importance immunization of rats with BP complexed to LPS results in the generation of cells in lymph nodes of these animals that suppress the mitogenic response of BP-immunized LNC and also SpC in mixed lymphocyte cultures. The suppressive effect of these cells in mixed lymphocyte culture reaction was found specifically in response to BP and to a lesser extent to LPS in LNC. SpC of BP-LPS immunized animals did not suppress the proliferative response to SpC of BP-immunized animals. Treatment of these LNC with antithymocyte serum and complement abolished this suppressive effect of LNC, suggesting that the immunoregulatory cells in LNC of BP-LPS immunized animals are suppressor T lymphocytes. The parallel between the in vitro induction of suppressor T lymphocytes in the draining LNC and the function of LPS in the development of EAE in Lewis rats suggests a possible immunologic significance of the effect.