Lymph Nodes Of Lewis Rats Research Articles

In Vitro Expansion of Regulatory T-Cells in the Rat

Introduction: CD4+CD25highFoxP3+ regulatory T-cells (Tregs) are of central importance in the immunological adaptation process following transplantation. Transfusing Tregs in order to induce tolerance represents a new therapeutical attempt to reduce or completely replace immunosuppressive treatment after transplantation. Thus, transplantation experiments in animal models (e.g. rat) currently are conducted to optimize this approach. However, the existing human protocols for in vitro expansion of Tregs cannot be applied directly transferred due to species specific differences. The aim of this study hence was the establishment of a method to expand rat regulatory T-cells from very small samples which would enable subsequent in vivo transplantation experiments - even using autologous cells. Methods: CD4+CD25high Tregs were isolated from lymph nodes of Lewis rats via cell sorting. Tregs were then cultured in RPMI-based medium in the presence of mAb to CD3 and CD28. Under the influence of different additional stimuli such as supplementation of interleukin-2 (IL-2), proliferation rates and viability of the cells were investigated. The influence of the applied culture conditions on the suppressive activity and phenotype of expanded Tregs was examined. Results: Following 7/14 days of expansion, the addition of IL-2 (n=15) led to approximately 3.3±3.1/3.4±2.2-fold expansion of Tregs, retaining a viability of 74.1±13.9%/32.2±8.9%, respectively. When replacing IL-2 with supernatant of a ConA-stimulated splenocyte culture (n=13), Tregs showed increased expansion rates of 11.4±8.8/8.3±4.2-fold (day 7/14) but at the same time a decrease in viability was evident (65.2±14.8% /24.7±10.4). Therefore the combination of both stimuli was tested as a third group (n=9), thus enhancing proliferation (9.4±3.5-fold) and cell viability (77.5±11.0%) which, in addition, remained stable until culture day 14 (9.8±7.4-fold expansion, 30.1±12.0% viability). The use of ConA-supernatant led to decreased FoxP3-expression as measured on day 7 (IL-2: 73.5±9.4%, ConA-supernatant: 27.6±2.2%, alternating stimuli: 43.4±22.5%) but interestingly, Tregs of all groups were functional and efficiently suppressed expansion of responder T-cells (IL-2: 94.1%, ConA-supernatant: 84.0%, alternating stimuli: 93.2%). Conclusion: The expansion of regulatory T-cells in the rat is significantly more difficult than in humans. However, the newly established protocol (IL-2 + supernatant of ConA-stimulated splenocytes) allows the efficient expansion of Tregs which maintain their strong suppressive functionality despite the partial loss of FoxP3-expression.

Expression of chemokine and receptors in Lewis rats with experimental autoimmune anterior uveitis

The purpose of this study is to investigate the sequential expression of certain chemokines and chemokine receptors in the iris–ciliary body and popliteal lymph nodes of Lewis rats and, thus, to establish their roles in experimental autoimmune anterior uveitis. Uveitis was induced with the injection of melanin-associated antigen intraperitoneally and into the left foot. The clinical severity of the uveitis was scored. At defined time points, CC chemokines (monocyte chemoattractant protein-1, macrophage inflammatory protein-1, and regulated-upon-activation normal T-cell expressed and secreted), CXC chemokines (interferon γ-inducible protein-10, stromal-derived factor-1, and interleukin-8), and receptor (CCR2, CCR3, CCR5, CXCR1, CXCR2, CXCR3, and CXCR4) mRNA expression were semiquantified by using a reverse-transcriptase reaction followed by polymerase chain reaction. The concentrations of macrophage inflammatory protein-1 and regulated-upon-activation normal T-cell expressed and secreted in aqueous humor were determined by means of enzyme-linked immunosorbent assay. Levels of monocyte chemoattractant protein-1, macrophage inflammatory protein-1 and interferon γ-inducible protein-10 started increasing before the clinical onset of disease; these might have been involved in the initial recruitment of inflammatory cells. The level of regulated-upon-activation normal T-cell mRNA, however, started rising concurrently with the onset of clinical disease, suggesting that this chemokine may exert amplifying role in generating uveitis. Stromal-derived factor-1 exhibited an early and high level of expression with the increase of cognate receptor, CXCR4, indicating that stromal-derived factor-1 plays a role in either promoting angiogenesis or attracting for T-cells. Instead of upregulation like other chemokine receptors, interleukin-8 receptors, CXCR1and CXCR2, mRNA could not be detected in accord with the increase of interleukin-8. These findings appeared that downregulation of chemokine receptors on neutrophils may make themselves less respond to interleukin-8 and subsequently lead to decreased recruitment of neutrophils into the iris–ciliary body. In addition, the expression of chemokine receptors in popliteal lymph nodes were earlier than those in the iris–ciliary body. This sequence of expression may reflect the process of T lymphocytes maturation and differentiation. Monocyte chemoattractant protein-1 protein was immunohistologically detected in the ciliary epithelium and infiltrating leukocytes. The above results suggest that chemokines, which act on T cells and monocytes, are sequentially upregulated during the clinical course of experimental autoimmune anterior uveitis, and thus, may contribute to the pathogenesis of acute anterior uveitis.

In vivo activity of leflunomide: pharmacokinetic analyses and mechanism of immunosuppression.

Leflunomide is an experimental drug with demonstrated ability to prevent and reverse acute allograft and xenograft rejection. The two biochemical activities reported for the active metabolite of leflunomide, A77 1726, are inhibition of tyrosine phosphorylation and inhibition of dihydroorotate dehydrogenase, an enzyme necessary for de novo pyrimidine synthesis. These activities can be distinctly separated in vitro by the use of uridine, which reverses the anti-proliferative effects of A77 1726 caused by inhibition of de novo pyrimidine synthesis. We report the effect of uridine on the in vivo immunosuppressive activities of leflunomide. We first quantified the serum levels of A77 1726, the active metabolite of leflunomide, after a single treatment of leflunomide (5, 15, and 35 mg/kg). Additionally, we quantified the levels of serum uridine and of nucleotide triphosphates in the liver, spleen, and lymph nodes of Lewis rats after the administration of a single dose of uridine (500 mg/kg; i.p.). Lewis rats heterotopically transplanted with brown Norway or Golden Syrian hamster hearts were treated for 50 or 75 days with leflunomide (5, 15, and 35 mg/kg/day; gavage) alone or in combination with uridine (500 mg/ kg/day; i.p.). Hematocrits were determined and the levels of alloreactive or xenoreactive immunoglobulin (Ig)M and IgG were determined by flow cytometric analysis. The allograft and xenografts, small bowel, liver, kidney, and spleen were subjected to pathological examination. A linear relationship was observed between the serum A77 1726 concentrations in Lewis rats and the dose of leflunomide administered. Peak A77 1726 concentrations were 20.9, 71.8 and 129.3 mg/l (77.5, 266.1 and 478.8 microM) for the 5, 15, and 35 mg/kg doses of leflunomide, respectively. The concentration of uridine in the serum of normal Lewis rats is 6.5 microM; after i.p. administration of 500 mg/kg uridine, the serum uridine concentrations peaked at 384.1 microM in 15-30 min. The rapid elimination of uridine was not reflected in the lymphoid compartments, and the pharmacokinetics of pyrimidine nucleotides in the spleen resembled that of A77 1726. This dose of uridine, when administered daily (500 mg/kg/day, i.p.), weakly antagonized the immunosuppressive activities of leflunomide (5, 15, and 35 mg/kg/day) in the allotransplantation model. In contrast, in the xenotransplantation model, the same concentration of uridine completely antagonized the immunosuppressive activities of low-dose leflunomide (15 mg/kg/day) and partially antagonized the immunosuppressive activities of high-dose leflunomide (35 mg/kg/day). Toxicities associated with high-dose leflunomide (35 mg/kg/day) were anemia, diarrhea, and pathological changes in the small bowel and liver. These toxicities were significantly reduced by uridine co-administration. These studies reveal that the blood levels of A77 1726 in Lewis rats satisfy in vitro requirements for both inhibition of de novo pyrimidine synthesis and protein tyrosine kinase activity. Our data also illustrate that the in vivo mechanism of immunosuppression by leflunomide is complex and is affected by at least the following four factors: type and vigor of the immune response, availability of uridine for salvage by proliferating lymphocytes, species being investigated, and concentration of serum A77 1726.

Autoreactive CD4- CD8- alpha beta T cells to vaccinate adjuvant arthritis.

Studies suggested that experimental autoimmune diseases can effectively be prevented and treated by application of normal autoreactive T cells or autoreactive T cells in an attenuated form. In this study, several autoreactive CD4- CD8- T-cell clones (A2, A6, and A13 cells) were isolated for the first time from the draining lymph nodes of Lewis rats with adjuvant arthritis (AA). Surprisingly, intraperitoneal inoculation with A13 cells, but not A2 or A6 cells protected rats from AA both clinically and histologically. It was demonstrated that A13 cells were CD4- CD8- alpha beta T cells, and showed proliferative responses to irradiated syngeneic spleen cells (antigen-presenting cells; APC). Interestingly, A13 cells proliferated against concanavalin A (Con A) and staphylococcal enterotoxin B (SEB), but did not show any proliferation to Mycobacterium tuberculosis (Mt), or its 65 000 MW heat-shock protein (HSP). Rats protected from AA by inoculation with A13 cells showed a specific anti-idiotypic delayed-type hypersensitivity reaction compared with other autoreactive T cells (A2 or A6 cells). These findings demonstrate that AA can be suppressed by autoreactive CD4- CD8- alpha beta T cells, and these cells may be used as therapeutic agents in experimental autoimmunity.

Arthritis by autoreactive T cell lines obtained from rats after injection of intestinal bacterial cell wall fragments

T cell lines (B13, B19) were isolated from the lymph nodes of Lewis rats 12 days after an arthritogenic injection of cell wall fragments of Eubacterium aerofaciens (ECW), a major resident of the human intestinal flora. These cell wall fragments consist of peptidoglycan polysaccharide complexes (PPC). The cell lines that bear the helper phenotype were arthritogenic in knee or ankle joints upon intravenous injection into irradiated Lewis recipients. B13 was, however, not arthritogenic in irradiated F344 recipients that are largely RT1 identical. The arthritis induced in the knee joints of the irradiated Lewis rats was clearly shown by a 99mtechnetium-pertechnetate scanning technique and was confirmed histologically. In vitro the cell lines showed a proliferative response after stimulation with syngeneic spleen cells alone. The proliferation was significantly higher when bacterial PPC, isolated in soluble form from normal feces or ileostomy fluid were added. Recognition by B13 appeared to be MHC class II restricted. These results show that autoreactive T cell lines can be isolated from rats after injection of bacterial cell wall antigens and that these cell lines can be arthritogenic. This suggests a role for autoreactive T cells in the induction of bacterial cell wall arthritis and might give a clue for the arthritogenic properties of the normal human intestinal flora.

T cell receptor β-chain usage in experimental autoimmune uveoretinitis

Genomic rearrangements to the T-cell receptor (TCR) Vβ8 gene locus were examined in T cells derived from the lymph nodes of Lewis rats immunized with either S-Antigen or peptides derived from interphotoreceptor retinoid binding protein (IRBP). The cells used in these studies are from T-cell lines that have been selected by several cycles of antigen/IL-2 stimulations, or clones isolated from these lines. No apparent rearrangement of the Vβ8 gene was observed by Southern analysis, suggesting that if indeed there are T cells using Vβ8 gene elements they represent small proportions of the cells in these T-cell lines that induce EAU (uveitogenic T cells) and that the lines may consist of large numbers of clones. On the other hand, we have demonstrated Vβ8 gene expression in uveitogenic T-cell populations by Northern analysis and by polymerase chain reaction (PCR). Although Vβ8 gene transcripts were detectable in pathogenic, but not in non-pathogenic, T-cell lines using a Vβ8 cDNA probe, RNA from pathogenic T cell lines did not hybridize to another probe specific for rat Vβ8.2. Taken together, these results suggest that, unlike the T-cell lines that mediate experimental allergic encephalomyelitis (EAE), some T-cell lines that induce EAU do not predominantly express Vβ8.2 gene but other member(s) of the Vβ8 family.

Tin compounds inhibit the plasma cell response to metallic tin

Injection of metallic tin powder causes intense proliferation of plasma cells in draining lymph nodes of Lewis rats. Pretreatment orally with soluble tin salts prevents this response to subsequently injected metallic tin. In the present work, pretreatment with tin salts by parenteral injection was just as effective as addition to the drinking water. This new approach made the following experiments possible. Poorly soluble tin compounds were found to be inhibitory when injected parenterally. Tin salts injected parenterally into one of two rats joined in parabiotic union prevented the plasma cell response to metallic tin in both parabionts. The transfer of the inhibitory effect via the cross-circulating blood represents significant progress toward understanding the mechanisms involved. The evidence suggests the possibility that tin salts elicit an intermediary substance or process that is responsible for inhibition of the plasma cell response to metallic tin.

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 × 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 O×8. 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.

Cardiovascular pathology induced by passive transfer of splenic cells from syngeneic rats treated with T-2 toxin

Mononuclear cells were separated from spleens and lymph nodes of Lewis rats treated 5 weeks previously with repeated small doses of T-2 toxin or solvent only. The cells from each site were then injected intraperitoneally into 4 groups of syngeneic Lewis rats. The animals injected with spleen cells from T-2 toxin-treated donors developed marked cardiovascular changes which were similar to those due to T-2 toxin itself. Rats injected with lymph node cells as well as those given each kind of cell from solvent-treated donors showed only occasional mild vascular changes. The changes seen after splenic cell transfer may be due to superinduction of interleukin 2 by T-2 toxin.

HgCl2 induces nonspecific immunosuppression in Lewis rats.

Brown-Norway (BN) rats injected with HgCl2 have been previously shown to develop a variety of autoimmune abnormalities. The susceptibility of BN rats is genetically controlled, and Lewis rats bearing a different RT1 haplotype are resistant. It will be shown in the present study that the number of MRC OX-8+ (suppressor/cytotoxic) cells increases in the spleen and lymph nodes of Lewis rats injected with HgCl2. The responsiveness to T cell mitogens and to alloantigens is concomitantly inhibited. Spleen cells from Lewis rats injected with HgCl2 fail to induce a local graft-vs.-host reaction. Data presented show that MRC OX-8+ cells are involved in the immunosuppression in Lewis rats treated with HgCl2. Furthermore, lymph node cells and MRC OX-8+ cells from these rats are able to inhibit the normal mixed lymphocyte reaction indicating that suppression is active. Thus, HgCl2 is able to trigger immune dysregulation leading either to autoimmunity or to immunosuppression depending upon the genetic background of the rat strain tested.

Regulation of experimental allergic encephalomyelitis: Part 4. Further characterization of postrecovery suppressor cells

Suppressor cells that regulate experimental allergic encephalomyelitis (EAE) are present in lymph nodes of Lewis rats that have recovered from the disease, as demonstrated by adoptive transfer of suppression for at least 108 days following challenge of donor rats with myelin basic protein (BL) in complete Freund's adjuvant (CFA). These suppressor lymph node cells (LNC) inhibit the occurrence of active, but not passive EAE in recipients, suggesting that they function early in the inductive phase of the response to BP. The suppressor cells are nylon-adherent, and suppression can be partially abrogated by depletion of B cells with anti-immunoglobulin (Ig) and complement (C). Serum from recovered donors has, at best, only a modest suppressive effect when transfused into syngeneic recipients. These findings suggest that the B cells do not mediate suppression of EAE via production of blocking antibody, but may function directly, perhaps by triggering T cells to become suppressors cells.

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.

The role of macrophages in the activation of T lymphocytes by concanavalin A. III. Macrophage-independent activation.

AbstractLymphocytes from the mesenteric lymph nodes of Lewis rats were depleted of macrophages by absorption for 60 min at 37°C on 0.1‐mm glass beads in the presence of 30% fetal calf serum. Lymphocytes which passed the column (total recovery 30‐50%) contained less than 0.2% macrophages, as assessed by neutral red ingestion or nonspecific esterase staining. At cell densities which allowed optimal proliferation of concanavalin A (Con A)‐stimulated, untreated lymphocytes (2 × 106 cells/ml), macrophage‐depleted lymphocytes were completely incapable of initiating DNA synthesis. The proliferative response to Con A was fully restored by adding back macrophages obtained from the peritoneal cavity. The help of macrophages was optimal at concentrations of 0.8 × 105 cells/ml, which corresponds to about 4% of lymphocytes. Restitution of the mitotic response of macrophage‐depleted lymphocytes was also achieved by raising cell densities to 5 × 106/ml. At this cell density, addition of macrophages was without any effect. Early events in lymphocyte activation, such as the increase of the incorporation of 14C‐oleate into membrane phospholipids after 4 h stimulation, was identical in the presence or absence of macrophages independent of the cell density ranging between 1 × 106 and 10 × 106 cells/ml. Similarly, the Con A‐stimulated early increase in the incorporation of [3H]uridine into RNA was indistinguishable in macrophage‐containing or ‐depleted lymphocytes. At low cell densities, only macrophage‐containing lymphocytes proceeded to increase RNA synthesis, until S‐phase, whereas in the absence of macrophages RNA synthesis approached background values after 40 h of Con A stimulation. Macrophage‐depleted lymphocytes, too, proceeded with an increase in RNA synthesis, when stimulated at high cell densities. The data suggest that macrophages are not required for the initiation of lymphocyte activation. Their role in supporting mitosis may be the contribution of a second signal close to the G1/S boundary extending the size of the proliferating population.

The Role of Macrophages in the Activation of T-Lymphocytes by Concanavalin A I. Macrophages Support Proliferation after Commitment of Lymphocytes

T lymphocytes require the presence of small numbers of macrophages to enter mitosis when activated by mitogens such as concanavalin A. The aim of this study was to decide when macrophages may operate in the course of activation. Lymphocytes from the mesenteric lymph nodes of Lewis rats were depleted of macrophages by adherence on 0.1 mm glass bead columns. Lymphocytes in the column effluent, between 40 and 50 percent of the cells originally placed on the column, contained less than 0.2 percent macrophages as determined by uptake of neutral red. Macrophage-depleted lymphocytes were completely unresponsive to stimulation with con A or PHA to enter DNA synthesis. The proliferative response to con A was completely restored by adding macrophages elicited intraperitoneally by various means. Addition of an optimal number of macrophages after 24 hrs of con A stimulation of macrophage-depleted lymphocytes also supported DNA synthesis, although to only 40-60% when compared to DNA synthesis in the continuous presence of macrophages, with a peak response temporarily identical to cultures where macrophages were present from the beginning. After 24 hrs of con A stimulation all lymphocytes were committed for mitogenesis, as removal of surface bound con A by a-methyl mannoside had no effect when added at this time. Substitution of macrophages after 24 hrs of con A stimulation in the presence of α-methyl mannoside resulted in an identical induction of DNA synthesis as in the absence of this hapten sugar. The data fit a hypothesis that initiation of lymphocyte activation with con A does not require the presence of macrophages. Macrophages may deliver a second signal, which is independent of the binding of the mitogen to these helper cells, and which supports induction of DNA replication after lymphocytes are committed for mitogenesis.

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 X 10(-3) M p-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.

Baisc protein hydrolysis in lymphocytes of Lewis rats with experimental allergic encephalomyelitis.

Lymphocytes from lymph nodes of Lewis rats with acute experimental allergic encephalomyelitis (EAE) contain high amounts of acid and neutral proteinases which hydrolyze myelin basic protein. The activity at neutral pH is also expressed by whole lymphocytes in isotonic medium, with about 50% more activity released by homogenization. Neutral proteinase activity in lymphocytes increases with the onset of acute EAE while the activity of those from Freund's adjuvant-injected controls increases somewhat later. The total neutral proteinase activity appears to be membrane-bound, most likely in the lysosomes, but half the total was associated with the nuclear fraction. The basic protein proteinase was compared with an enzyme described earlier, especially active toward polylysine, and some differences were noted. It appears that two enzymes may be present in lymphocytes which hydrolyze basic protein at a neutral pH. An increase in neutral proteinase activity was observed in some, but not all, lymphocyte preparations from patients in various stages of multiple sclerosis. The finding that whole activated lymphocytes are capable of hydrolyzing basic protein suggests that these cells which are believed to be precursors of mononuclear cells migrating into the central nervous system may be active agents in the early stages of myelin dissolution in experimental allergic encephalomyelitis. At present, such a mechanism is only theoretical, and the possibility that activated lymphocytes may be a factor in demyelination in multiple sclerosis is even more speculative.