Arthur A. Vandenbark

Experimental stroke induces massive, rapid activation of the peripheral immune system

Clinical experimental stroke induces injurious local brain inflammation. However, effects on the peripheral immune system have not been well characterized. We quantified mRNA and protein levels for cytokines, chemokines, and chemokine receptors (CCR) in brain, spinal cord, peripheral lymphoid organs (spleen, lymph node, blood, and cultured mononuclear cells from these sources), and blood plasma after reversible middle cerebral artery occlusion (MCAO) or sham treatment in male C57BL/6 mice. Middle cerebral artery occlusion induced a complex, but organ specific, pattern of inflammatory factors in the periphery. At both 6 and 22 h after MCAO, activated spleen cells from stroke-injured mice secreted significantly enhanced levels of TNF-α, IFN-γ, IL-6, MCP-1, and IL-2. Unstimulated splenocytes expressed increased chemokines and CCR, including MIP-2 and CCR2, CCR7 & CCR8 at 6 h; and MIP-2, IP-10, and CCR1 & CCR2 at 22 h. Also at 22 h, T cells from blood and lymph nodes secreted increased levels of inflammatory cytokines after activation. As expected, there were striking proinflammatory changes in postischemic brain. In contrast, spinal cord displayed suppression of all mediators, suggesting a compensatory response to intracranial events. These data show for the first time that focal cerebral ischemia results in dynamic and widespread activation of inflammatory cytokines, chemokines, and CCR in the peripheral immune system.

Cutting Edge: Estrogen Drives Expansion of the CD4+CD25+ Regulatory T Cell Compartment

CD4+CD25+ regulatory T cells are crucial to the maintenance of tolerance in normal individuals. However, the factors regulating this cell population and its function are largely unknown. Estrogen has been shown to protect against the development of autoimmune disease, yet the mechanism is not known. We demonstrate that estrogen (17-β-estradiol, E2) is capable of augmenting FoxP3 expression in vitro and in vivo. Treatment of naive mice with E2 increased both CD25+ cell number and FoxP3 expression level. Further, the ability of E2 to protect against autoimmune disease (experimental autoimmune encephalomyelitis) correlated with its ability to up-regulate FoxP3, as both were reduced in estrogen receptor α-deficient animals. Finally, E2 treatment and pregnancy induced FoxP3 protein expression to a similar degree, suggesting that high estrogen levels during pregnancy may help to maintain fetal tolerance. In summary, our data suggest E2 promotes tolerance by expanding the regulatory T cell compartment.

Low-Dose Estrogen Therapy Ameliorates Experimental Autoimmune Encephalomyelitis in Two Different Inbred Mouse Strains

It has been proposed that homeostatic levels of estrogen can enhance female susceptibility to autoimmunity, whereas the heightened levels of estrogen associated with pregnancy are protective. This hypothesis was tested using the mouse model of experimental autoimmune encephalomyelitis (EAE). Diestrus (<100 pg/ml in serum) levels of 17β-estradiol were found to significantly reduce the clinical manifestations of active EAE in both male and female mice. Estriol was also effective but at doses below those previously established for pregnancy. The reduction in disease severity was accompanied by a coincident reduction in the number and size of inflammatory foci in the CNS of estrogen (17β-estradiol or estriol)-treated mice. Recipients of encephalitogenic T cells from low-dose estrogen-treated mice developed less severe paralysis than mice receiving T cells from placebo-treated mice. A modest shift in Th1/Th2 balance suggested that low dose estrogen therapy could bias the immune reaction toward a protective anti-inflammatory cytokine response. However, estrogen treatment at the onset of active EAE failed to reduce disease severity, a result that is consistent with the hypothesis that naive cells are more sensitive to sex hormones than differentiated effector cells. These data suggest that treatment with low doses of estrogen can reduce the capacity of developing myelin-reactive T cells to initiate disease and challenges the idea that increased susceptibility to autoimmunity in females is dependent on homeostatic levels of estrogen.

Decreased FOXP3 Levels in Multiple Sclerosis Patients

Autoimmune diseases such as multiple sclerosis (MS) may result from the failure of tolerance mechanisms to prevent expansion of pathogenic T cells. Our study is the first to establish that MS patients have abnormalities in FOXP3 message and protein expression levels in peripheral CD4+CD25+ T cells (Tregs) that are quantitatively related to a reduction in functional suppression induced during suboptimal T‐cell receptor (TCR) ligation. Of importance, this observation links a defect in functional peripheral immunoregulation to an established genetic marker that has been unequivocally shown to be involved in maintaining immune tolerance and preventing autoimmune diseases. Diminished FOXP3 levels thus indicate impaired immunoregulation by Tregs that may contribute to MS. Future studies will evaluate the effects of therapies known to influence Treg cell function and FOXP3 expression, including TCR peptide vaccination and supplemental estrogen.

Splenic atrophy in experimental stroke is accompanied by increased regulatory T cells and circulating macrophages

Induction of stroke not only produces local ischemia and brain damage, but also has profound effects on peripheral immune responses. In the current study, we evaluated effects on spleen and blood cells 4 days after stroke induction. Surprisingly, there was a less inflammatory cytokine profile in the middle cerebral artery occlusion-affected right brain hemisphere at 96 h compared with earlier time points. Moreover, our results demonstrate that stroke leads to splenic atrophy characterized by a reduction in organ size, a drastic loss of splenocyte numbers, and induction of annexin V+ and TUNEL+ cells within the spleen that are in the late stages of apoptosis. The consequence of this process was to reduce T cell proliferation responses and secretion of inflammatory cytokines, resulting in a state of profound immunosuppression. These changes produced a drastic reduction in B cell numbers in spleen and blood, and a novel increase in CD4+FoxP3+ regulatory T cells. Moreover, we detected a striking increase in the percentage of nonapoptotic CD11b+ VLA-4-negative macrophages/monocytes in blood. Immunosuppression in response to brain injury may account for the reduction of inflammatory factors in the stroke-affected brain, but also potentially could curtail protective immune responses in the periphery. These findings provide new evidence to support the contention that damage to the brain caused by cerebral ischemia provides a powerful negative signal to the peripheral immune system that ultimately induces a drastic state of immunosuppression caused by cell death as well as an increased presence of CD4+FoxP3+ regulatory T cells.

T- and B-cell-deficient mice with experimental stroke have reduced lesion size and inflammation

Stroke induction in immunologically competent mice not only produces local ischemia and brain damage, but also induces early inflammatory changes in brain and peripheral immune responses. Although immune elements clearly are activated after brain vascular occlusion, the relative contribution of T and B lymphocytes to the developing lesion has not been quantified. We evaluated effects 22 h after middle cerebral artery occlusion (90 mins) on histologic injury and peripheral immune activation in severe combined immunodeficient (SCID) mice lacking T and B cells. Cortical and total infarct volumes were strikingly reduced in male SCID mice (n = 14, 33 ± 4% of contralateral cortex, n = 10, 52 ± 3% of contralateral hemisphere) versus immunologically intact C57BL/6 mice (wild type, n = 9, 57 ± 5% of contralateral cortex, 57 ± 4% of contralateral hemisphere) (P < 0.01). Striatal infarction was not altered (77 ± 7% of contralateral striatum in SCID, 84 ± 7% in wild type), suggesting that the core of the evolving ischemic lesion was not impacted by lack of T and B cells. As expected, inflammatory factors from immune cells in ischemic SCID brains were essentially absent, with the exception of interleukin-1β increase in both SCID and wild type tissue. Spleen cell numbers were low in SCID mice, but were further reduced 22 h after stroke, with substantial reduction in most inflammatory factors except for increased expression of interferon-γ and macrophage inflammatory protein (MIP)-2. These data quantify the damaging effect of T and B lymphocytes on early, evolving ischemic brain injury, and further implicate interleukin-1β in brain and interferon-γ and MIP-2 in spleen as inflammatory factors produced by cells other than T and B cells.

Treatment of multiple sclerosis with T–cell receptor peptides: Results of a double–blind pilot trial

A T–cell receptor (TCR) peptide vaccine from the Vβ5.2 sequence expressed in multiple sclerosis (MS) plaques and on myelin basic protein (MBP)–specific T cells boosted peptide–reactive T cells in patients with progressive MS. Vaccine responders had a reduced MBP response and remained clinically stable without side effects during one year of therapy, whereas nonresponders had an increased MBP response and progressed clinically. Peptide–specific T helper 2 cells directly inhibited MBP–specific T helper 1 cells in vitro through the release of interleukin–10, implicating a bystander suppression mechanism that holds promise for treatment of MS and other autoimmune diseases.

Depressed lymphocyte responsiveness in autistic children.

Although there are associations linking autism with prenatal rubella, cytomegalovirus, syphilis, and varicella, the etiology of the autistic state remains obscure. Host defense against the etiologic agents postulated to be responsible for the autism-associated syndromes is believed to be primarily of the cell-mediated type. In this preliminary study, cellular immune function was assessed in vitro by phytohemagglutinin (PHA)stimulation of lymphocyte cultures. Twelve autistic children and 13 control subjects were compared. The autistic group exhibited a depressed lymphocyte transformation response to PHA when compared to the control subjects (p<.01).

Functional assay for human CD4+CD25+ Treg cells reveals an age-dependent loss of suppressive activity.

CD4+CD25+ regulatory T cells (Treg cells) prevent T cell‐mediated autoimmune diseases in rodents. To develop a functional Treg assay for human blood cells, we used FACS‐ or bead‐sorted CD4+CD25+ T cells from healthy donors to inhibit anti‐CD3/CD28 activation of CD4+CD25− indicator T cells. The data clearly demonstrated classical Treg suppression of CD4+CD25− indicator cells by both CD4+CD25+high and CD4+CD25+low T cells obtained by FACS or magnetic bead sorting. Suppressive activity was found in either CD45RO− (naive) or CD45RO+ (memory) subpopulations, was independent of the TCR signal strength, required cell–cell contact, and was reversible by interleukin‐2 (IL‐2). Of general interest is that a wider sampling of 27 healthy donors revealed an age‐ but not gender‐dependent loss of suppressive activity in the CD4+CD25+ population. The presence or absence of suppressive activity in CD4+CD25+ T cells from a given donor could be demonstrated consistently over time, and lack of suppression was not due to method of sorting, strength of signal, or sensitivity of indicator cells. Phenotypic markers did not differ on CD4+CD25+ T cells tested ex vivo from suppressive vs. nonsuppressive donors, although, upon activation in vitro, suppressive CD4+CD25+ T cells had significantly higher expression of both CTLA‐4 and GITR than CD4+CD25− T cells from the same donors. Moreover, antibody neutralization of CTLA‐4, GITR, IL‐10, or IL‐17 completely reversed Treg‐induced suppression. Our results are highly consistent with those reported for murine Treg cells and are the first to demonstrate that suppressive activity of human CD4+CD25+ T cells declines with age. Published 2003 Wiley‐Liss, Inc.

Frequency of T cells specific for myelin basic protein and myelin proteolipid protein in blood and cerebrospinal fluid in multiple sclerosis

T cell sensitization to two myelin components, myelin basic protein (MBP) and myelin proteolipid protein (PLP), may be important to the pathogenesis of multiple sclerosis (MS). Using the limiting dilution assay, we demonstrated that the blood of MS patients had an increased frequency of MBP-reactive T cells compared with normal subjects and patients with other neurological diseases (OND) and rheumatoid arthritis. There was no difference in T cell frequency to a synthetic peptide, PLP139-151, or Herpes simplex virus. Within cerebrospinal fluid (CSF), 37% of IL-2/IL-4-reactive T cell isolates from MS patients responded either to MBP or PLP139-151 while only 5% of similar isolates from OND patients responded to these myelin antigens. The mean relative frequency of MBP-reactive T cells within CSF from MS patients was significantly higher than that of OND patients (22 x 10(-5) cells versus 1 x 10(-5) cells) and was similar to that of MBP reactive T cells within the central nervous system of rats with experimental autoimmune encephalomyelitis. These results lend new support to the hypothesis that myelin-reactive T cells mediate disease in MS.