Immune Regulatory Mechanisms Influence Early Pathology in Spinal Cord Injury and in Spontaneous Autoimmune Encephalomyelitis
2005; Elsevier BV; Volume: 166; Issue: 6 Linguagem: Inglês
10.1016/s0002-9440(10)62485-6
ISSN1525-2191
AutoresMaria Cecília Garibaldi Marcondes, Gláucia C. Furtado, Allen Wensky, Maria A. Curotto de Lafaille, Howard S. Fox, Juan J. Lafaille,
Tópico(s)Autoimmune and Inflammatory Disorders Research
ResumoInjuries to the central nervous system (CNS) trigger an inflammatory reaction with potentially devastating consequences. In this report we compared the characteristics of the inflammatory response on spinal cord injury (SCI) caused by a stab wound between the T7 and T9 vertebrae and spontaneous experimental autoimmune encephalomyelitis (EAE). SCI and EAE were compared in two types of myelin basic protein Ac1-11-specific T-cell receptor transgenic mice: T/R+ mice harbor regulatory T cells, and T/R− mice lack regulatory T cells. Our results show that 8 days after SCI, T/R− mice developed a strong T-cell infiltrate in the spinal cord, with remarkable down-modulation of CD4 expression that was accompanied by a local increase in Mac-3+ and F4/80+ reactivity and diffuse local and distal astrogliosis. In contrast, T/R+ mice exhibited a modest increase in CD4+ cells localized to the site of injury, without CD4 down-modulation; focal astrogliosis was restricted to the site of the lesion, although Mac-3+ and F4/80+ cells were also present. Similarly to T/R− mice that underwent SCI, T cells displaying down-modulated CD4 expression were found in the CNS of older T/R− mice afflicted by spontaneous EAE. Overall, our results suggest that common mechanisms regulate T-cell accumulation in CNS lesions of different causes, such as mechanic lesion or autoimmune-mediated damage. Injuries to the central nervous system (CNS) trigger an inflammatory reaction with potentially devastating consequences. In this report we compared the characteristics of the inflammatory response on spinal cord injury (SCI) caused by a stab wound between the T7 and T9 vertebrae and spontaneous experimental autoimmune encephalomyelitis (EAE). SCI and EAE were compared in two types of myelin basic protein Ac1-11-specific T-cell receptor transgenic mice: T/R+ mice harbor regulatory T cells, and T/R− mice lack regulatory T cells. Our results show that 8 days after SCI, T/R− mice developed a strong T-cell infiltrate in the spinal cord, with remarkable down-modulation of CD4 expression that was accompanied by a local increase in Mac-3+ and F4/80+ reactivity and diffuse local and distal astrogliosis. In contrast, T/R+ mice exhibited a modest increase in CD4+ cells localized to the site of injury, without CD4 down-modulation; focal astrogliosis was restricted to the site of the lesion, although Mac-3+ and F4/80+ cells were also present. Similarly to T/R− mice that underwent SCI, T cells displaying down-modulated CD4 expression were found in the CNS of older T/R− mice afflicted by spontaneous EAE. Overall, our results suggest that common mechanisms regulate T-cell accumulation in CNS lesions of different causes, such as mechanic lesion or autoimmune-mediated damage. Cell infiltration and accumulation are features of the tissue response to distinct stimuli, such as traumatic injury, infections, or under some circumstances, self-components. Whatever leads to the presence of leukocytes in tissues, the result ranges from the development of chronic inflammation to a successful resolution. In organs such as the brain, even small lesions can cause important physiological consequences, physical impairment, and behavioral alterations, according to the severity of the response and its localization. Mechanical central nervous system (CNS) lesions can cause primary damage to axons and result in inflammatory reactions1Ghirnikar RS Lee YL Eng LF Inflammation in traumatic brain injury: role of cytokines and chemokines.Neurochem Res. 1998; 23: 329-340Crossref PubMed Scopus (240) Google Scholar, 2Popovich PG Wei P Stokes BT Cellular inflammatory response after spinal cord injury in Sprague-Dawley and Lewis rats.J Comp Neurol. 1997; 377: 443-464Crossref PubMed Scopus (786) Google Scholar that lead to migration of several types of leukocytes, including T lymphocytes. T lymphocytes are activated when they contact the cognate MHC-peptide complexes. Additional co-stimulatory molecules such as B7 family members participate triggering and modulating the T-cell response.3Lenschow DJ Walunas TL Bluestone JA CD28/B7 system of T cell costimulation.Annu Rev Immunol. 1996; 14: 233-258Crossref PubMed Scopus (2375) Google Scholar, 4Sharpe AH Freeman GJ The B7-CD28 superfamily.Nat Rev Immunol. 2002; 2: 116-126Crossref PubMed Scopus (1414) Google Scholar, 5Okazaki T Iwai Y Honjo T New regulatory co-receptors: inducible co-stimulator and PD-1.Curr Opin Immunol. 2002; 14: 779-782Crossref PubMed Scopus (198) Google Scholar Most T cells that migrate into the CNS after lesion do not recognize CNS antigens.6Brocke S Gijbels K Allegretta M Ferber I Piercy C Blankenstein T Martin R Utz U Karin N Mitchell D Veromaa T Waisman A Gaur A Conlon P Ling N Fairchild PJ Wraith DC O'Garra A Fathman CG Steinman L Treatment of experimental encephalomyelitis with a peptide analogue of myelin basic protein.Nature. 1996; 379: 343-346Crossref PubMed Scopus (371) Google Scholar However, in some experimental models it was shown that bystander activation of CD8+ T cells could have a role secondary to the specific Ag recognition, causing tissue damage.7Haring JS Pewe LL Perlman S Bystander CD8 T cell-mediated demyelination after viral infection of the central nervous system.J Immunol. 2002; 169: 1550-1555PubMed Google Scholar, 8Haring JS Perlman S Bystander CD4 T cells do not mediate demyelination in mice infected with a neurotropic coronavirus.J Neuroimmunol. 2003; 137: 42-50Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar Because CNS-specific T cells are rare, the role of CNS-specific T cells that infiltrate the CNS as a consequence of traumatic injury is not completely understood. To address this question, we took advantage of a mouse model in which a large number of the circulating T cells are specific for myelin basic protein (MBP).9Lafaille JJ Nagashima K Katsuki M Tonegawa S High incidence of spontaneous autoimmune encephalomyelitis in immunodeficient anti-myelin basic protein T cell receptor transgenic mice.Cell. 1994; 78: 399-408Abstract Full Text PDF PubMed Scopus (489) Google Scholar This experimental system allowed us to address the role of myelin-specific T cells on CNS stab wound lesions and correlate it with development of other CNS pathologies, such as experimental autoimmune encephalomyelitis (EAE). Mice harboring large number of cells expressing T-cell receptor (TCR) specific for MBP Ac1-11 were previously generated.9Lafaille JJ Nagashima K Katsuki M Tonegawa S High incidence of spontaneous autoimmune encephalomyelitis in immunodeficient anti-myelin basic protein T cell receptor transgenic mice.Cell. 1994; 78: 399-408Abstract Full Text PDF PubMed Scopus (489) Google Scholar These animals, designated T/R+ mice, have a very high frequency of T lymphocytes that recognize the MBP Ac1-11 peptide presented by the I-Au MHC class II molecule. Despite the fact that the vast majority of CD4+ T cells in T/R+ mice are MBP-specific and immunocompetent, T/R+ animals do not develop any spontaneous CNS pathology. The presence of functional RAG genes in T/R+ mice allows some degree of recombination of endogenous TCR genes, generating a small number of lymphocytes with a diverse recognition repertoire. When T/R+ mice were bred with RAG1-deficient mice, generating T/R− mice, 100% of the animals developed EAE spontaneously.9Lafaille JJ Nagashima K Katsuki M Tonegawa S High incidence of spontaneous autoimmune encephalomyelitis in immunodeficient anti-myelin basic protein T cell receptor transgenic mice.Cell. 1994; 78: 399-408Abstract Full Text PDF PubMed Scopus (489) Google Scholar Although the number of anti-MBP CD4+ T cells is essentially the same between T/R+ and T/R− mice, a comparison of lymphocytic populations between T/R+ and T/R− mice indicated that T/R− mice lack: 1) a small population of αβ T cells expressing TCR encoded by the endogenous (nontransgenic) α and β loci, including some CD8+ T cells; 2) γδ T cells; and 3) B cells, which are present in normal numbers in T/R+ mice. Because of the RAG mutation, T/R− mice harbor exclusively MBP-specific T lymphocytes. In subsequent studies we crossed T/R+ mice with β2-microglobulin-deficient mice, which lack CD8+ T cells, with μMT mice, which lack mature B cells, with TCRδ gene-deficient mice, which lack γδ-T cells, and with TCRα and TCRβ gene-deficient mice, which can express transgenic MBP-specific TCR chains but not endogenous TCRα and TCRβ chains, respectively.10Olivares-Villagomez D Wang Y Lafaille JJ Regulatory CD4(+) T cells expressing endogenous T cell receptor chains protect myelin basic protein-specific transgenic mice from spontaneous autoimmune encephalomyelitis.J Exp Med. 1998; 188: 1883-1894Crossref PubMed Scopus (261) Google Scholar Our results, as well as those of others, showed that, insofar as prevention of spontaneous EAE, only CD4+ T cells expressing endogenous αβ TCRs were essential.10Olivares-Villagomez D Wang Y Lafaille JJ Regulatory CD4(+) T cells expressing endogenous T cell receptor chains protect myelin basic protein-specific transgenic mice from spontaneous autoimmune encephalomyelitis.J Exp Med. 1998; 188: 1883-1894Crossref PubMed Scopus (261) Google Scholar, 11Van de Keere F Tonegawa S CD4(+) T cells prevent spontaneous experimental autoimmune encephalomyelitis in anti-myelin basic protein T cell receptor transgenic mice.J Exp Med. 1998; 188: 1875-1882Crossref PubMed Scopus (138) Google Scholar Ensuing studies determined that not all αβ CD4+ T cells are protective against EAE, ruling out competition models, and eventually supporting a key role for the specificity of the TCRs expressed by regulatory T cells.12Olivares-Villagomez D Wensky AK Wang Y Lafaille JJ Repertoire requirements of CD4+ T cells that prevent spontaneous autoimmune encephalomyelitis.J Immunol. 2000; 164: 5499-5507PubMed Google Scholar However, spontaneous EAE in T/R− mice could be suppressed by the presence of regulatory CD4+ T cells. For the regulatory function, the expression of the CD25 marker, which is traditionally associated with regulatory T cells,13Furtado GC Olivares-Villagomez D Curotto de Lafaille MA Wensky AK Latkowski JA Lafaille JJ Regulatory T cells in spontaneous autoimmune encephalomyelitis.Immunol Rev. 2001; 182: 122-134Crossref PubMed Scopus (145) Google Scholar is not necessary, although an interleukin (IL)-2-dependent in vivo conversion of CD25− regulatory T cells into CD4+CD25+ T cells correlates with the acquisition of regulatory functions and Foxp3 expression.14Furtado GC Curotto de Lafaille MA Kutchukhidze N Lafaille JJ Interleukin-2 signaling is required for CD4+ regulatory T cell function.J Exp Med. 2002; 196: 851-857Crossref PubMed Scopus (515) Google Scholar, 15Curotto de Lafaille MA Lino AC Kutchukhidze N Lafaille JJ CD25− T cells generate CD25+Foxp3+ regulatory T cells by peripheral expansion.J Immunol. 2004; 173: 7259-7268PubMed Google Scholar We and others also transferred total spleen cells or purified CD4+ cells from wild-type syngeneic animals and showed that they could confer protection against the development of spontaneous EAE when administered before the onset of disease.10Olivares-Villagomez D Wang Y Lafaille JJ Regulatory CD4(+) T cells expressing endogenous T cell receptor chains protect myelin basic protein-specific transgenic mice from spontaneous autoimmune encephalomyelitis.J Exp Med. 1998; 188: 1883-1894Crossref PubMed Scopus (261) Google Scholar, 11Van de Keere F Tonegawa S CD4(+) T cells prevent spontaneous experimental autoimmune encephalomyelitis in anti-myelin basic protein T cell receptor transgenic mice.J Exp Med. 1998; 188: 1875-1882Crossref PubMed Scopus (138) Google Scholar Therefore, a regulatory pool of cells is present in normal individuals, which is contained within the population of αβ CD4+ cells. The role of the regulatory mechanisms on CNS lesions is an extremely relevant point in addition to the role of CNS-specific effector T cells, leading to insights into the evolution and repair of CNS lesions. To further understand the regulation of CNS lesions, T/R+ and T/R− mice were subjected to a stab wound made between the T7 and T9 vertebrae in the spinal cord. The frequency of antigen (Ag)-specific cells and their activation status was determined in animals of different ages. The phenotype of the infiltrating lymphocytes was compared to spontaneous EAE. We found significant differences in the extent of the inflammatory response in animals that harbor (T/R+) or do not harbor (T/R−) regulatory T cells. These differences parallel those found in spontaneous EAE. We conclude that T-cell responses, both harmful and protective, follow common pathways regardless of the triggering signal being a mechanical lesion or a spontaneously developing autoimmune disease. MBP Ac1-11-specific transgenic TCR mice (T/R+ and T/R−) on B10.PL genetic background (H-2u) were previously described.9Lafaille JJ Nagashima K Katsuki M Tonegawa S High incidence of spontaneous autoimmune encephalomyelitis in immunodeficient anti-myelin basic protein T cell receptor transgenic mice.Cell. 1994; 78: 399-408Abstract Full Text PDF PubMed Scopus (489) Google Scholar To generate T/R− mice, T/R+ mice were crossed to RAG-1 knockout mice.16Mombaerts P Iacomini J Johnson RS Herrup K Tonegawa S Papaioannou VE RAG-1-deficient mice have no mature B and T lymphocytes.Cell. 1992; 68: 869-877Abstract Full Text PDF PubMed Scopus (2364) Google Scholar All animals were maintained under SPF conditions at the Skirball Institute Animal Facility, New York University Medical Center. EAE was scored as described by Baron and colleagues17Baron JL Madri JA Ruddle NH Hashim G Janeway Jr, CA Surface expression of alpha 4 integrin by CD4 T cells is required for their entry into brain parenchyma.J Exp Med. 1993; 177: 57-68Crossref PubMed Scopus (792) Google Scholar: level 1, limp tail; level 2, weak or partial leg paralysis; level 3, total hind leg paralysis; level 4, hind leg paralysis and weak or partial front leg paralysis; level 5, moribund (immediately sacrificed). Protocols were approved by New York University Institutional Animal Care and Use Committee. The mice were anesthetized with an intraperitoneal injection of 0.2 ml of a 2.5% solution of 2,2,2-tri-bromo-ethanol (Sigma, St. Louis, MO) in distilled water. The level of anesthesia was evaluated by tests of cornea reflex to peripheral pain stimulus. The surgical area was shaved and disinfected with iodide. The animals were placed on a heated surface, for a cutaneous incision on the dorsal area between T1 and T12. The muscle on the T8 region was exposed to allow the visualization of the vertebrae under a scope. The localization of T8 was made based on the fact that T13 corresponds to the vertebrae associated to the last rib, with retrograde count. A laminectomy was performed and a 22½-gauge needle was inserted for 2 mm at a 45-degree angle. After suture, the animals were maintained under heat. All procedures were approved by the New York University Institutional Animal Care and Use Committee. Mice were anesthetized with Metofane (Metoxifluorane; Mallinckrodt Veterinary, Inc., Mundelein, IL), to obtain a 50-μl blood aliquot from orbital plexus. After a 10-minute perfusion with phosphate-buffered saline (PBS) containing 5 mmol/L ethylenediamine tetraacetic acid, the vertebral column was removed and divided in four segments with equivalent length: R1 (from the cervical C1 to the thoracic T2), R2 (from T3 to T9), R3 (from T10 to the lumbar L4), and R4 (from L5 to Co3). Dissected segments were placed in separate wells of a 48-well plate. Blood cells were submitted to the same treatment for control. To each well, 100 μl of a 10-mg/ml type VI collagenase was added. The samples were incubated for 1 hour at 37°C, additionally disrupted with a pipette, and passed through a 45-μm nylon mesh. The volume of each sample was completed to 860 μl to which 530 μl of an 89% Percoll-saline solution was added. The samples were centrifuged at 300 × g for 30 minutes. The pellet was resuspended in 100 μl of PBS containing 5% fetal calf serum and 0.1% sodium azide (staining buffer), washed twice in the same buffer, and incubated 45 minutes with the respective labeled antibodies for flow cytometric analysis of phenotype (2.5 μg/106 cells). After this period, the cells were washed twice and resuspended in propidium iodide for exclusion of dead cells and debris. Cells isolated as described (2 × 105 to 10 × 105) were stained with 50-μl mixtures of antibodies diluted according to a previous titration in staining buffer (Hanks' balanced salt solution with 2% fetal calf serum and 0.01% NaN3). The antibodies used for the staining were anti-CD3 (Pharmingen, San Diego, CA), anti-CD4 (Pharmingen), and an anti-MBP TCR clonotypic monoclonal antibody, 3H12. 3H12 is a mouse IgM generated in our laboratory as described;10Olivares-Villagomez D Wang Y Lafaille JJ Regulatory CD4(+) T cells expressing endogenous T cell receptor chains protect myelin basic protein-specific transgenic mice from spontaneous autoimmune encephalomyelitis.J Exp Med. 1998; 188: 1883-1894Crossref PubMed Scopus (261) Google Scholar it recognizes the transgenic MBP-specific T cells while staining virtually no cells in B10.PL nontransgenic littermates.10Olivares-Villagomez D Wang Y Lafaille JJ Regulatory CD4(+) T cells expressing endogenous T cell receptor chains protect myelin basic protein-specific transgenic mice from spontaneous autoimmune encephalomyelitis.J Exp Med. 1998; 188: 1883-1894Crossref PubMed Scopus (261) Google Scholar Isotype controls (Pharmingen) were also used. The cells were then processed through a FACScalibur flow cytometer before analysis of data with CellQuest software (Becton-Dickinson Immunocytometry Systems, San Jose, CA). Tissues were fixed in 10% formalin, embedded in paraffin, and cut into 5-μm sections. Sections were serially assembled on slides, labeled according to animal numbers and without reference to experimental groups, to avoid biased analysis. After hematoxylin and eosin (H&E) staining, sections from each tissue block were examined microscopically. Serial sections were immunohistochemically stained. Briefly, 5-mμ sections mounted on charged slides were deparaffinized and hydrated. Endogenous peroxidase was blocked by a 30-minute treatment with 0.3% H2O2 in 100% P.A. methanol. Nonspecific binding was blocked with 5 g/L casein in PBS solution, pH 7.4, in a PBS-humidified chamber. The slides were incubated overnight with the primary antibody anti-GFAP (DAKO, Carpinteria, CA), or anti-Mac 3 (Pharmingen), or anti-F4/80 (clone X2; kindly donated by Dr. Nora Sarvetnick, the Scripps Research Institute, La Jolla, CA), diluted to 1.5 μg/ml in casein block solution, under low agitation, at 4°C. After that, slides were incubated for 1 hour with biotin-labeled anti-rat IgG (Vector Laboratories, Burlingame, CA), followed by a 30-minute incubation with streptavidin-peroxidase (Zymed Laboratories Inc., San Francisco, CA). Color was developed with NovaRed (Vector Laboratories), according to the manufacturer's instructions. Slides were counterstained with Gill's hematoxylin, rehydrated, mounted, and microscopically evaluated. Splenic cells were incubated with bead-labeled anti-CD4 antibodies (Miltenyi Biotec, Auburn, CA), according to the manufacturer's protocol, and passed through VarioMacs magnetic columns (Miltenyi Biotec) attached to a magnet. The purity of CD4-enriched and -depleted fractions was checked by fluorescence-activated cell sorting (FACS), before in vivo injection. One-week-old T/R− mice received intraperitoneally 2 × 107 total splenic cells, magnetically sorted CD4+ cells, or unbound, CD4-depleted fraction, from wild-type mice. At 4 to 5 weeks of age, circulating CD4+ 3H12− cells were quantified by FACS. At 4 or 5 weeks of age, SCI was performed as described. Normality and homogeneity of variances were verified by Tukey and Bartlett's test, respectively. Samples were compared using Kruskal-Wallis nonparametric analysis of variance, followed by Dunn's multiple comparisons. Statistical analysis was performed using GraphPad Prism Software (San Diego, CA). A stab wound spinal cord injury (SCI) was performed on the T7 to T9 vertebral segment in the spinal cord of T/R+ and T/R− animals. Both T/R+ and T/R− mice harbor a large number of MBP-specific T cells, but T/R+ mice also have a small population of T cells displaying capacity to prevent the occurrence of spontaneous EAE. In contrast, the T-cell repertoire in T/R− mice consists exclusively of MBP-specific T cells.9Lafaille JJ Nagashima K Katsuki M Tonegawa S High incidence of spontaneous autoimmune encephalomyelitis in immunodeficient anti-myelin basic protein T cell receptor transgenic mice.Cell. 1994; 78: 399-408Abstract Full Text PDF PubMed Scopus (489) Google Scholar, 10Olivares-Villagomez D Wang Y Lafaille JJ Regulatory CD4(+) T cells expressing endogenous T cell receptor chains protect myelin basic protein-specific transgenic mice from spontaneous autoimmune encephalomyelitis.J Exp Med. 1998; 188: 1883-1894Crossref PubMed Scopus (261) Google Scholar, 11Van de Keere F Tonegawa S CD4(+) T cells prevent spontaneous experimental autoimmune encephalomyelitis in anti-myelin basic protein T cell receptor transgenic mice.J Exp Med. 1998; 188: 1875-1882Crossref PubMed Scopus (138) Google Scholar, 13Furtado GC Olivares-Villagomez D Curotto de Lafaille MA Wensky AK Latkowski JA Lafaille JJ Regulatory T cells in spontaneous autoimmune encephalomyelitis.Immunol Rev. 2001; 182: 122-134Crossref PubMed Scopus (145) Google Scholar We compared the histopathology of T/R+ and T/R− mice in H&E-stained sections from the site of the lesion, and from the lumbar region, which is distant from the stab wound. Analysis of the site of the lesion showed remarkable differences between the two groups. Figure 1 shows representative 5-week-old animals from the T/R+ (A) and T/R− (B) groups, 8 days after a SCI. At this time point it was possible to distinguish between the two groups based on the density and distribution pattern of infiltrating cells. T/R− mice showed a much higher number of infiltrating cells with a focal as well as diffuse pattern of distribution, with a large area of lesion and edema, as opposed to T/R+ mice, which had a significantly lower density, smaller and at most focally restricted infiltrate. Overall, at 8 days after SCI, the T/R− mice developed a lesion with a larger number of cells and denser pattern of infiltration than did the T/R+ animals. Two additional types of analysis provided support for the conclusions reached on examination of the H&E-stained specimens: flow cytometry and immunohistochemistry. The infiltrating cells in both groups of transgenic animals as well as wild-type controls were characterized by flow cytometry, performed on cell suspensions from segments of the spinal cord. The initial characterization of cell populations was made using anti-CD4 and the anti-clonotypic antibody 3H12, which recognizes the MBP-specific TCR.10Olivares-Villagomez D Wang Y Lafaille JJ Regulatory CD4(+) T cells expressing endogenous T cell receptor chains protect myelin basic protein-specific transgenic mice from spontaneous autoimmune encephalomyelitis.J Exp Med. 1998; 188: 1883-1894Crossref PubMed Scopus (261) Google Scholar Figure 2 shows the percentage of CD4+ 3H12+ cells in the T7 to T9 segment of the spinal cord of representative 5-week-old animals from T/R− (A) and T/R+ (B) groups. Because all of the events from the T7 to T9 region were collected in the cytometer, it was possible to compare absolute and relative numbers of MBP-specific T lymphocytes, based on the number of events. Eight days after SCI of 5-week-old mice, there were 4600 ± 2100 MBP-specific T cells in the T7 to T9 region of T/R− mice, compared to only 296 ± 95 cells in T/R+ mice. Because there are exceedingly few 3H12+ cells in wild-type mice, in these animals we determined the infiltration by total CD4+ cells. This number was 25 ± 16 cells (n = 17, 7, and 4, for T/R−, T/R+, and wild-type mice, respectively). We also performed the SCI procedure in animals of different ages (4, 5, and 6 weeks old), and performed flow cytometry analysis of the MBP-specific T-cell infiltrate. These data are presented in Table 1.Table 1Percentage of MBP-Specific CD4+3H12+ T Cells in the T7–T9 and Segment of the Spinal Cord in T/R+ and T/R− Mice at Different Ages, 8 Days after SCI, or after Mock SurgeryAgeT7–T9LumbarNT/R+*For wild-type animals (NT/R+), the values correspond to the total CD4+ population. We do not show the frequency of CD4+ 3H12+ cells in wild-type mice because it is below the limit of sensitivity of the method, ie, there is no difference between 3H12-stained and unstained samples.T/R+†P < 0.05 between different groups at matching ages.T/R−†P < 0.05 between different groups at matching ages.NT/R+*For wild-type animals (NT/R+), the values correspond to the total CD4+ population. We do not show the frequency of CD4+ 3H12+ cells in wild-type mice because it is below the limit of sensitivity of the method, ie, there is no difference between 3H12-stained and unstained samples.T/R+T/R−Mock 4 weeks0.0 ± 0.00.02 ± 0.010.05 ± 0.030.01 ± 0.00.05 ± 0.050.065 ± 0.02 5 weeks0.0 ± 0.00.03 ± 0.020.18 ± 0.080.01 ± 0.010.11 ± 0.060.12 ± 0.05 6 weeks0.01 ± 0.010.07 ± 0.040.03 ± 0.010.0 ± 0.00.06 ± 0.060.11 ± 0.05SCI 8 days 4 weeks1.96 ± 0.40.17 ± 0.021.84 ± 0.040.43 ± 0.10.08 ± 0.070.08 ± 0.02 5 weeks1.24 ± 0.22.15 ± 1.2217.64 ± 3.76†P < 0.05 between different groups at matching ages.0.25 ± 0.150.44 ± 0.234.08 ± 1.23†P < 0.05 between different groups at matching ages. 6 weeks1.35 ± 0.33.16 ± 1.55†P < 0.05 between different groups at matching ages.21.39 ± 8.65†P < 0.05 between different groups at matching ages.0.30 ± 0.120.52 ± 0.26.13 ± 2.2†P < 0.05 between different groups at matching ages.The percentage of total CD4+ T cells in wild-type (NT/R+) animals, subjected to SCI or mock surgery, is also shown. The values are the average of six animals per group ± SD.* For wild-type animals (NT/R+), the values correspond to the total CD4+ population. We do not show the frequency of CD4+ 3H12+ cells in wild-type mice because it is below the limit of sensitivity of the method, ie, there is no difference between 3H12-stained and unstained samples.† P < 0.05 between different groups at matching ages. Open table in a new tab The percentage of total CD4+ T cells in wild-type (NT/R+) animals, subjected to SCI or mock surgery, is also shown. The values are the average of six animals per group ± SD. Table 1 and Figure 2 illustrate several important points. First, consistent with the histological analysis, the number of infiltrating T cells is significantly higher in T/R− mice than in T/R+ mice or wild-type mice subjected to SCI. The clear differences in the number (above) and frequency of infiltrating cells shown in Figure 2 8 days after SCI were, however, not apparent 48 hours after lesion (data not shown). Animals subjected to mock surgery did not show any appreciable inflammatory infiltrate (Table 1). Second, age-dependent differences in the accumulation of infiltrating lymphocytes were observed on SCI in T/R− mice. The T-cell infiltrate that followed the stab wound was, clearly, more intense in 5- and 6-week-old animals than in 4-week-old animals. This is particularly interesting given the fact that clinical manifestations of spontaneous EAE in T/R− mice do not occur before 6 weeks of age, despite the fact that large numbers of immune competent MBP-specific T cells circulate in T/R− mice since birth. Thus, massive accumulation of MBP-specific T cells in the CNS does not take place before 5 weeks of age even when a stab wound is produced. Third, although the spinal cords of T/R+ mice had much fewer infiltrating T cells than T/R− mice, after 5 weeks of age they harbored much more infiltrating T cells than wild-type B10.PL (NT/R+) mice (above, and Table 1). This is consistent with a previous report by Jones and co-workers18Jones TB Basso DM Sodhi A Pan JZ Hart RP MacCallum RC Lee S Whitacre CC Popovich PG Pathological CNS autoimmune disease triggered by traumatic spinal cord injury: implications for autoimmune vaccine therapy.J Neurosci. 2002; 22: 2690-2700PubMed Google Scholar comparing the same T/R+ mouse strain with wild-type mice. Fourth, in T/R+ mice, a relatively large proportion of 3H12-negative CD4+ T cells could be found in the spinal cord (Figure 2B, top left). 3H12-negative T cells in T/R+ mice express an array of endogenous TCR chains, mostly TCR α chains;10Olivares-Villagomez D Wang Y Lafaille JJ Regulatory CD4(+) T cells expressing endogenous T cell receptor chains protect myelin basic protein-specific transgenic mice from spontaneous autoimmune encephalomyelitis.J Exp Med. 1998; 188: 1883-1894Crossref PubMed Scopus (261) Google Scholar expression of endogenous TCR chains confers broad reactivity to the T cells that express them. As we showed previously,9Lafaille JJ Nagashima K Katsuki M Tonegawa S High incidence of spontaneous autoimmune encephalomyelitis in immunodeficient anti-myelin basic protein T cell receptor transgenic mice.Cell. 1994; 78: 399-408Abstract Full Text PDF PubMed Scopus (489) Google Scholar, 10Olivares-Villagomez D Wang Y Lafaille JJ Regulatory CD4(+) T cells expressing endogenous T cell receptor chains protect myelin basic protein-specific transgenic mice from spontaneous autoimmune encephalomyelitis.J Exp Med. 1998; 188: 1883-1894Crossref PubMed Scopus (261) Google Scholar the percentage of 3H12− CD4+ T cells does not exceed 5% of the total CD4+ cells in the periphery of T/R+ mice, but reaches values close to 30% in their injured spinal cord. Thus, it is likely that T cells with non-CNS specificity can be found among infiltrating T cells, in accordance with previously describe
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