John B. Hay

Chemokine receptor CCR7 required for T lymphocyte exit from peripheral tissues

Lymphocytes travel throughout the body to carry out immune surveillance and participate in inflammatory reactions. Their path takes them from blood through tissues into lymph and back to blood. Molecules that control lymphocyte recruitment into extralymphoid tissues are well characterized, but exit is assumed to be random. Here, we showed that lymphocyte emigration from the skin was regulated and was sensitive to pertussis toxin. CD4+ lymphocytes emigrated more efficiently than CD8+ or B lymphocytes. T lymphocytes in the afferent lymph expressed functional chemokine receptor CCR7, and CCR7 was required for T lymphocyte exit from the skin. The regulated expression of CCR7 by tissue T lymphocytes may control their exit, acting with recruitment mechanisms to regulate lymphocyte transit and accumulation during immune surveillance and inflammation.

Toluidine Blue Staining Identifies High-Risk Primary Oral Premalignant Lesions with Poor Outcome

There is a pressing need for the development of visual aids that will facilitate the detection of oral premalignant lesions (OPLs) with a high-risk of progression. Preliminary data suggest that toluidine blue stain may be preferentially retained by OPLs with high-risk molecular clones. In this study, we monitored OPLs from 100 patients without any history of oral cancer for an average of 44 months in order to evaluate the association of toluidine blue status with clinicopathologic risk factors, molecular patterns (microsatellite analysis on seven chromosome arms: 3p, 9p, 4q, 8p, 11q, 13q, and 17p) and outcome. Toluidine blue-positive staining correlated with clinicopathologic risk factors and high-risk molecular risk patterns. Significantly, a >6-fold elevation in cancer risk was observed for toluidine blue-positive lesions, with positive retention of the dye present in 12 of the 15 lesions that later progressed to cancer (P = 0.0008). This association of toluidine blue status with risk factors and outcome was evident even when the analysis was restricted to OPLs with low-grade or no dysplasia. Our results suggest the potential use of toluidine blue in identifying high-risk OPLs.

Drainage of CSF through lymphatic pathways and arachnoid villi in sheep: measurement of 125I-albumin clearance.

We investigated lymphatic drainage pathways of the central nervous system in conscious sheep and quantified the clearance of a cerebrospinal fluid (CSF) tracer into lymph and blood. In the first group of studies, 125I‐HSA was injected into the lateral ventricles of the brain or into lumbar CSF and after 6 h, various lymph nodes and tissues were excised and counted for radioactivity. Multiple lymphatic drainage pathways of cranial CSF existed in the head and neck region defined by elevated 125I‐HSA in the retropharyngeal/cervical, thymic, pre‐auricular and submandibular nodes. Implicated in spinal CSF drainage were mainly the lumbar and intercostal nodes. In a second group of experiments, multiple cervical vessels and the thoracic duct were cannulated and lymph diverted from the animals. Transport of tracer through arachnoid villi was taken from recoveries in venous blood. Following intraventricular administration, the 6 h recoveries of 125I‐HSA in the lymph (sum of cervical and thoracic duct) and blood were 8.2%± 3.0 and 12.5%± 4.5 respectively and at 22 h, 25.1%± 6.9 and 20.8%± 4.1 respectively. When 125I‐HSA was injected into lumbar CSF, the 6 h recoveries of tracer in thoracic duct and blood were 11.6%± 2.7 and 16.3%± 3.7 respectively. Total lymph and blood recoveries were not significantly different in any experiment. We conclude that the clearance of 125I‐HSA from the CSF is almost equally distributed between lymphatic and arachnoid villi pathways.

Determination of volumetric cerebrospinal fluid absorption into extracranial lymphatics in sheep

We estimated the volumetric clearance of cerebrospinal fluid (CSF) through arachnoid villi and extracranial lymphatics in conscious sheep. Catheters were inserted into both lateral ventricles, the cisterna magna, multiple cervical lymphatics, thoracic duct, and jugular vein. Uncannulated cervical vessels were ligated.125I-labeled human serum albumin (HSA) was administered into both lateral ventricles.131I-HSA was injected intravenously to permit calculation of plasma tracer loss and tracer recirculation into lymphatics. From mass balance equations, total volumetric absorption of CSF averaged 3.37 ± 0.38 ml/h, with 2.03 ± 0.29 ml/h (∼60%) removed by arachnoid villi and 1.35 ± 0.46 ml/h (∼40%) cleared by lymphatics. With projected estimates for noncannulated ducts, total CSF absorption increased to 3.89 ± 0.33 ml/h, with 1.86 ± 0.49 ml/h (48%) absorbed by lymphatics. Additionally, we calculated total CSF drainage to be 3.48 ± 0.52 ml/h, with 54 and 46% removed by arachnoid villi and lymphatics, respectively, using previously published mass transport data from our group. We employed estimates of CSF tracer concentrations that were extrapolated from relationships observed in the study reported here. We conclude that 40-48% of the total volume of CSF absorbed from the cranial compartment is removed by extracranial lymphatic vessels.We estimated the volumetric clearance of cerebrospinal fluid (CSF) through arachnoid villi and extracranial lymphatics in conscious sheep. Catheters were inserted into both lateral ventricles, the cisterna magna, multiple cervical lymphatics, thoracic duct, and jugular vein. Uncannulated cervical vessels were ligated. 125I-labeled human serum albumin (HSA) was administered into both lateral ventricles. 131I-HSA was injected intravenously to permit calculation of plasma tracer loss and tracer recirculation into lymphatics. From mass balance equations, total volumetric absorption of CSF averaged 3.37 +/- 0.38 ml/h, with 2.03 +/- 0.29 ml/h (approximately 60%) removed by arachnoid villi and 1.35 +/- 0.46 ml/h (approximately 40%) cleared by lymphatics. With projected estimates for noncannulated ducts, total CSF absorption increased to 3.89 +/- 0.33 ml/h, with 1.86 +/- 0.49 ml/h (48%) absorbed by lymphatics. Additionally, we calculated total CSF drainage to be 3.48 +/- 0.52 ml/h, with 54 and 46% removed by arachnoid villi and lymphatics, respectively, using previously published mass transport data from our group. We employed estimates of CSF tracer concentrations that were extrapolated from relationships observed in the study reported here. We conclude that 40-48% of the total volume of CSF absorbed from the cranial compartment is removed by extracranial lymphatic vessels.

The modulation of enhanced vascular permeability by prostaglandins through alterations in blood flow (hyperemia)

The enhanced vascular permeability induced by histamine or bradykinin in the skin of the guinea-pig and rabbit was significantly augmented by small amounts of prostaglandins of the E type. When injected alone these prostaglandins had little effect on vascular permeability. Furthermore, E type prostaglandins were found to be more potent at inducing hyperemia than either histamine or bradykinin. Prostaglandin F2α did not enhance the vascular permeability induced by histamine or bradykinin nor did it produce hyperemia in the skin. In the rat, prostaglandins alone enhanced vascular permeability but they also increased the effect of histamine, serotonin and bradykinin. Using85Sr-microspheres to measure blood flow a correlation was found between the degree of hyperemia produced by prostaglandins and the degree to which they augmented enhanced vascular permeability due to histamine, serotonin or bradykinin. Prostaglandins therefore can directly mimic the hyperemia of the inflammatory process and can also modulate the changes in vascular permeability caused by other mediators of inflammation.

Long-term tracking of lymphocytes in vivo: The migration of PKH-labeled lymphocytes

Studies of in vivo cell migration using cell markers such as 51Cr, 111In, FITC, or XRITC have been limited to short time periods due to the elution, toxicity, or rapid loss of label detectability. We have labeled sheep lymphocytes in vitro with PKH-2, a new fluorescent cell membrane label, and, after their intravenous injection back into donor sheep, have been able to detect them in efferent lymph, using flow cytometry, for longer than 38 days. The PKH-2-labeled lymphocytes migrated with similar kinetics, efficiency, and tissue specificity as lymphocytes labeled with cell markers used previously. PKH-2-labeled cells mediated graft versus host reactions indistinguishable from those mediated by unlabeled cells, and cell surface antigens were equally detectable on the surface of labeled and unlabeled lymphocytes. According to the slow, consistent loss of fluorescence intensity of the labeled cells in vivo, we predict that labeled lymphocytes could remain detectable by flow cytometry for greater than 7 weeks with the labeling protocol used in these experiments.

Raised intracranial pressure increases CSF drainage through arachnoid villi and extracranial lymphatics

We demonstrated previously that about one-half of cerebrospinal fluid (CSF) removed from the cranial vault was cleared by extracranial lymphatic vessels. In this report we test the hypothesis that lymphatic drainage of CSF increases as intracranial pressure (ICP) is elevated in anesthetized sheep. Catheters were inserted into both lateral ventricles, cisterna magna, cervical lymphatics, and jugular vein. A ventriculocisternal perfusion system was employed to regulate CSF pressures and to deliver a protein tracer (125I-labeled human serum albumin) into the CSF compartment. 131I-labeled human serum albumin was injected intravenously to permit calculation of plasma tracer loss and tracer recirculation into lymphatics. ICP was controlled by adjusting the height of the inflow reservoir and the cisterna magna outflow catheter appropriately. The experimental design consisted of a 3-h period of lower pressure followed by a 3-h period of higher pressure in the same animal (10-20 or 20-30 cmH2O). We determined that incremental changes in ICP were associated with higher CSF transport through lymphatic and arachnoid villi routes in all eight animals tested (P = 0.004).We demonstrated previously that about one-half of cerebrospinal fluid (CSF) removed from the cranial vault was cleared by extracranial lymphatic vessels. In this report we test the hypothesis that lymphatic drainage of CSF increases as intracranial pressure (ICP) is elevated in anesthetized sheep. Catheters were inserted into both lateral ventricles, cisterna magna, cervical lymphatics, and jugular vein. A ventriculocisternal perfusion system was employed to regulate CSF pressures and to deliver a protein tracer (125I-labeled human serum albumin) into the CSF compartment.131I-labeled human serum albumin was injected intravenously to permit calculation of plasma tracer loss and tracer recirculation into lymphatics. ICP was controlled by adjusting the height of the inflow reservoir and the cisterna magna outflow catheter appropriately. The experimental design consisted of a 3-h period of lower pressure followed by a 3-h period of higher pressure in the same animal (10-20 or 20-30 cmH2O). We determined that incremental changes in ICP were associated with higher CSF transport through lymphatic and arachnoid villi routes in all eight animals tested ( P = 0.004).

Chemoattractant Receptors and Lymphocyte Egress from Extralymphoid Tissue: Changing Requirements during the Course of Inflammation

Memory/effector T cells traffic efficiently through extralymphoid tissues, entering from the blood and leaving via the afferent lymph. During inflammation, T cell traffic into the affected tissue dramatically increases; however, the dynamics and mechanisms of T cell exit from inflamed tissues are poorly characterized. In this study, we show, using both a mouse and a sheep model, that large numbers of lymphocytes leave the chronically inflamed skin. Many T cells capable of producing IFN-γ and IL-17 also entered the draining afferent lymph, demonstrating that memory/effector T cells egress from sites of inflammation. Whereas efficient egress from acutely inflamed skin required lymphocyte-expressed CCR7, chronic inflammation promoted significant CCR7-independent exit as well. Lymphocyte exit at late time points of inflammation was sensitive to pertussis toxin but was only partially affected by the drug FTY720, implying the contribution of alternative chemoattractant receptors other than spingosine 1-phosphate receptor 1. Our data show that CCR7 is an important receptor for lymphocyte egress from both resting and inflamed extralymphoid tissues, but that alternative exit receptors come into play during chronic inflammation.

The release of phospholipase A2 from aggregated platelets and stimulated macrophages of sheep

Abstract Phospholipase A2 release from platelets and macrophages was assayed by means of a modified, rapid bioassay based on the hydrolysis of E. coli membranes. Enzyme activity was found in the supernatants of both washed, aggregated platelets and concanavalin A stimulated macrophages from afferent lymph of sheep. Phospholipase A2 may be related to the vasoactive moiety secreted by antigen or mitogen stimulated macrophages.

Effect of cold preservation on lymphocyte migration into peripheral nerve allografts in sheep

Lymphocyte migration into fresh and preserved peripheral nerve allografts was quantitated to assess the effect of cold preservation and freeze-thawing pretreatment on the local immunological response to nerve allografts. Out-bred ewes received multiple 1.5-cm sub cutaneous heterotopic peroneal nerve autografts, fresh allografts, and pretreated allografts, implanted within the same recipient. Lymphocyte migration was studied at 7 days by injecting autologous 111indium-labeled lymphocytes intravenously. After 3 hr of recirculation, lymphocyte migration into graft tissue was quantitated by a gamma counter (cpm/g, mean ± SEM). Lymphocyte traffic into fresh nerve allografts (21,623 ± 3783) increased an average 9.4-fold over the autograft value (2918 ± 377, P<0.04). Histologic studies illustrated a marked lymphocytic infiltrate of CD4+ and CD8+ cells and enhanced class I and II MHC expression in fresh allografts, but not in autografts. Short-term cold preservation, for 6 and 12 hr (5°C), enhanced lymphocyte entry into pretreated allograft tissue. Conversely, cold preservation for longer periods (1 and 3 weeks) dramatically reduced lymphocyte migration to values below corresponding autograft levels (783±100 and 1,252 ± 120, respectively, P<0.01). A comparable reduction in