Characterization of white matter damage in animal models of multiple sclerosis by magnetization transfer ratio and quantitative mapping of the apparent bound proton fraction f.

Quantitative magnetization transfer magnetic resonance imaging (qMT-MRI) can be used to improve detection of white matter tissue damage in multiple sclerosis (MS) and animal models thereof. To study the correlation between MT parameters and tissue damage, the magnetization transfer ratio (MTR), the parameter f* (closely related to the bound proton fraction) and the bound proton transverse relaxation time T(2B) of lesions in a model of focal experimental autoimmune encephalomyelitis (EAE) were measured on a 7T animal scanner and data were compared with histological markers indicative for demyelination, axonal density, and tissue damage. A clear spatial correspondence was observed between reduced values of MTR and demyelination in this animal model. We observed two different levels of MTR and f* reduction for these lesions. One was characterized by a pronounced demyelination and the other corresponded to a more severe loss of the cellular matrix. Changes in f* were generally more pronounced than those of MTR in areas of demyelination. Moreover, a reduction of f* was already observed for tissue where MTR was virtually normal. No changes in T(2B) were observed for the lesions. We conclude that MTR and qMT mapping are efficient and reliable readouts for studying demyelination in animal models of MS, and that the analysis of regional f* might be even superior to the analysis of MTR values. Therefore, quantitative mapping of f* from human brains might also improve the detection of white matter damage in MS.

Lymph, lymphocytes, and lymphatics.

This is a summary of recent developments regarding the role of the lymphatic system in immune responses. Emphasis is on physiological considerations from experiments in sheep. Cell- and tissue-specific lymphocyte traffic patterns measured over several days are considered. Particular attention is given to recent data on the relationship between the central nervous system and the lymphatic system, to cell labeling in situ, and to the entry of immune cells into afferent lymph from the interstitial tissues.

Tissue specificity of lymphocyte migration into sheep gingival tissue.

T cells show a bias in their migration pathways: some migrate preferentially to peripheral lymph nodes, some to mucosal tissues and some to peripheral tissues such as skin. The aim here was to determine the types of T cells that migrate preferentially into inflamed gingival tissue and compare this migration to that found in inflamed subcutaneous and mucosal tissues. The experiments were designed so that the simultaneous 3 h localization of two, differentially radiolabelled, lymphocyte populations (subcutaneously and mucosally derived) into sites of purified protein derivative/bacillus Calmette-Guerin-induced, delayed-type hypersensitivity, inflammatory lesions in skin, bowel and gingiva in the sheep model could be compared. The relative migration of two populations in each of the tissues was expressed as a ratio of the radioactivity of intestinal/subcutaneous lymphocytes recovered from that tissue. From nine experiments, the ratios [mean+/-S.E.M. (n)] for skin, bowel and gingiva were 0.53+/-0.02 (84), 1.98+/-0.11 (85), and 0.73+/-0.05 (29), respectively. These findings suggest that inflammation in skin and gingiva favoured the localization of subcutaneously derived lymphocytes (ratio significantly <1, P<0.025), while in bowel, the localization of intestinally derived lymphocytes was favoured (ratio significantly >1, P<0.025). Statistical analysis demonstrated that the relative localization of the two lymphocyte populations to the gingival lesions differed significantly from that for inflamed skin and bowel lesions (P<0.05). When tumour necrosis factor-alpha was used as a non-antigenic inflammatory agent to induce lymphocyte migration into skin and gingiva, a similarly greater increase in the localization of subcutaneously derived lymphocytes was detected, but the relative localization of lymphocytes was not significantly different between the two tissues. Therefore, it appears that there is tissue specificity in the migration of lymphocytes into the inflamed gingival tissues and that antigen is required for distinct tissue-specific lymphocyte traffic to occur.

Epinephrine causes a reduction in lymph node cell output in sheep.

The lymphatic system has a critical role in the return of fluids, proteins, and cells to the circulatory system. However, the effects of stress, including exercise, on this system have not been adequately studied. We investigated the effect of a physiological dose (1 mg) of epinephrine (Epi) on lymph flow, cell concentration, and lymphocyte subsets in efferent subcutaneous lymph in sheep. Blood leukocyte numbers, differential, lymphocyte subsets, and blood and lymph pools of lymphocytes were determined simultaneously. A significant acute increase in lymph flow was followed by a post-injection decrease in flow and cellular output. No changes in lymphocyte subsets or pools of lymphocytes were seen in either blood or lymph. The timing of elevated plasma and lymph concentrations of Epi and norepinephrine (NE) corresponded with the increased lymph flow. In conclusion, Epi injection caused no change in lymphocyte subset distribution, leukocyte concentration, or pools of lymphocytes. A decrease in lymph flow and cellularity was documented post-injection, indicating that lymphatic tissue has no role in the leukocytosis seen after Epi injection. Lymphocyte retention by lymph nodes, however, may contribute to post-injection lymphopenia.

Cervical lymph cannulation to investigate the efflux and effects of intracerebroventricular cytokine infusions.

It is well documented that there is communication between the cerebral spinal fluid (CSF) and cervical lymphatics. Recently, it has been demonstrated that tumor necrosis factor alpha (TNF-alpha) introduced into the CSF appears in the cervical lymph. However, the functional significance of this is less clear. Here we describe a protocol to quantitate the efflux of TNF-alpha from the CSF into cervical lymph. In addition, we describe a methodology to examine the effects of an intracerebroventricular (i.c.v.) infusion of TNF-alpha on lymph volume, cellularity and cell phenotype. While TNF-alpha was recovered in the cervical lymph following infusion of 125-I labeled TNF-alpha, the dosage of TNF-alpha used in this study had no effect on cervical lymph flow, cellularity or cell subsets. This protocol can be used to study the efflux of i.c.v. injected macromolecules and their effects on lymphocytes in cervical lymph and the regional lymph nodes.

Intracerebroventricular infusions of TNF-alpha preferentially recruit blood lymphocytes and induce a perivascular leukocyte infiltrate.

Tumour necrosis factor (TNF)-alpha is important in several central nervous system (CNS) inflammatory diseases, however, its role in the recruitment of leukocytes into the cerebral spinal fluid (CSF) and CNS is incompletely understood. Therefore, we examined the effect of intracerebroventricular (icv) and parenchymal infusions of TNF-alpha on the type of leukocyte, the pool and subset of lymphocytes recruited into CSF and brain parenchyma. Parenchymal injections of 500 ng of recombinant human TNF-alpha did not induce inflammation, whereas an icv infusion of TNF-alpha caused CSF leuckocytosis and a perivascular infiltrate. Twenty-four hours after the icv infusion neutrophils predominated, with CD4+ T cells being the major lymphocyte subset in CSF. By 48 h lymphocytes were the dominant cell type with CD8+ cells surpassing CD4+ cells in both the CSF and the perivascular infiltrate. The labeled recirculating lymphocyte pool prevailed in normal CSF, but after the infusion of TNF-alpha, the blood pool of lymphocytes was preferentially recruited. These results have implications for the immune surveillance of the CNS.

Splenectomy selectively affects the distribution and mobility of the recirculating lymphocyte pool.

The spleen plays a major role in immune surveillance, but the impact that splenectomy exerts on the immune competence of an individual is not fully resolved. Here we show that neonatal splenectomy in sheep does not abrogate the development of a large, nonrecirculating pool of lymphocytes and that it has no effect on the acquisition of a normal blood lymphocyte profile. Splenectomy did, however, result in a significant decrease in blood residency time of recirculating lymphocytes and in an enhanced accumulation of recirculating lymphocytes in lymph nodes. Furthermore, nonrecirculating peripheral blood lymphocytes were less likely to migrate to the lung, possibly because of saturation of the marginal pool by recirculating lymphocytes. Although splenectomy has little effect on the development or distribution of lymphocyte subsets in blood and lymph, it has marked effects on the rate of recirculation of lymphocytes, which may have significant implications for peripheral immune surveillance in patients who undergo splenectomy.

A role for lymphatic endothelium in the sequestration of recirculating gamma delta T cells in TNF-alpha-stimulated lymph nodes.

TNF-alpha is one of the most potent immunoregulatory molecules in vivo. In addition to important regulatory effects, it is also a potent inducer of extravascular lymphocyte infiltration. To examine the dynamic changes that are induced in local lymphocyte migration through regional lymph nodes following TNF-alpha injection, we used a protocol of direct lymphatic cannulation to quantitatively and qualitatively examine the traffic of lymphocytes through regional lymph nodes. We observed that local TNF-alpha injection reduced the output of lymphocytes from lymph nodes up to 90% within 6-10 h following stimulation. TNF-alpha also altered the specificity of migration of lymphocyte traffic through subcutaneous lymph nodes. In addition to the decreased output, phenotypic analysis demonstrated decreases in the concentration of gamma delta T cells by up to 30% following TNF-alpha injection. Histological examination showed that the gamma delta T cells were found in close association with VCAM-1-expressing cells in TNF-stimulated lymph nodes, at least some of which appeared to be lymphatic endothelium. These data indicate that TNF-alpha is capable of altering the number and specificity of lymphocytes recirculating through stimulated lymph nodes by selectively altering the entry of lymphocytes into the efferent lymphatics of inflamed lymph nodes in vivo.

The traffic of resting lymphocytes through delayed hypersensitivity and chronic inflammatory lesions: a dynamic equilibrium.

This essay is designed as a partial summary of the work of several students and colleagues from our laboratory. For the most part the experimental data have been published and this seminar represents an attempt to summarize, integrate and speculate on this work. In some situations the speculation is rather unrestrained and it is hoped that it will provoke discussion, controversy and better future experiments. Reference is made to the original articles and to recent reviews. In addition, since we have had the advantage of reading the other contributions to this volume, there is considerable reference to other chapters. We and others have argued in other publications that it is imperative to understand the normal physiological traffic of lymphocytes before one can adequately interpret data describing lymphocyte migration through pathological tissues. It has been useful to compare data derived from traffic through lymph nodes because there is considerable information on the individual lymph node with respect to blood-lymphocyte delivery and blood flow, prenodal input via peripheral lymphatics and, particularly in sheep, in the numbers and phenotypic analysis of the lymphocytes exiting lymph nodes in postnodal or efferent lymph (see Young, this volume). We consider the terms prenodal for afferent and post-nodal for efferent to be synonomous. In this volume, a significant contribution has been made by Cahill et al which describes the astonishing degree of lymphocyte traffic which occurs in fetal life prior to antigenic challenge. At the other extreme, in disease states, there is often profound activation of lymphocytes. This is most apparent in viral infections like HIV and SIV (Rosenberg et al, this volume) and also in the various models of diseases such as EAE (Hickey and Kulidjian et al, this volume). It is a central tenet of our paper that resting and activated lymphocyte migration need to be considered separately and that they are very different.

Cerebral spinal fluid lymphocytes are part of the normal recirculating lymphocyte pool.

We have investigated the migration of lymphocytes from blood into the central nervous system (CNS) under normal physiological conditions. Using sheep as our model, we simultaneously sampled blood, lymph and cerebral spinal fluid (CSF). Normal, nonactivated, recirculating lymphocytes can migrate into the CSF in similar concentrations as found in subcutaneous lymph and there is no difference in the temporal appearance between them. Lymphocytes infused into the CNS could be found in cervical lymph nodes. These data suggest that lymphocytes found in the CNS are part of the recirculating lymphocyte pool and do not require activation to enter the CSF.

The use of the lipophilic fluorochrome CM-DiI for tracking the migration of lymphocytes.

In this study we examined the new cell dye CM-DiI for tracking the migration of lymphocytes from blood to lymph. This lipophilic marker intercalates in the plasma membrane like the PKH dyes and older DiI derivatives. The stability and intensity of staining achieved with these dyes is better than most other fluorochromes or radioisotopes, yet they are poorly soluble in aqueous solutions, which can make staining difficult, and they are not fixable in tissue sections. CM-DiI is reported to have increased water solubility and it can be fixed using traditional aldehyde fixatives, making it feasible to detect labeled cells in histological sections. To determine the suitability of CM-DiI as a lymphocyte marker, a labeling protocol was developed. We tested the ability of stained cells to recirculate in vivo. Following the intravenous injection of CM-DiI positive cells, their recovery in lymph over 40 h was comparable to that of cells labeled with other fluorochromes or radioisotopes. The kinetics of recirculation were also very similar, as labeled cells were detectable in lymph within 4 h of injection, and the peak percentage of labeled cells in lymph was generally observed between 20-30 h. We also confirmed that CM-DiI is retained in the lymphocyte membrane following routine paraffin processing. Thus CM-DiI does not appear to alter the process of lymphocyte recirculation, and it should be a useful marker for tracking these cells.