The protective effects of omega-6 fatty acids in experimental autoimmune encephalomyelitis (EAE) in relation to transforming growth factor-beta 1 (TGF-beta1) up-regulation and increased prostaglandin E2 (PGE2) production.

Polyunsaturated fatty acids are known to affect the immune response and administration of the omega-6 fatty acid linoleic acid has been reported to be beneficial in multiple sclerosis (MS) and EAE. In this study we have investigated the effects of oral feeding of plant lipid rich in the omega-6 fatty acid gamma-linolenic acid from Borago officinalis on acute and relapse disease and the immune response in EAE using SJL mice. EAE was induced by an encephalitogenic peptide (92-106) of myelin oligodendrocyte glycoprotein (MOG), and mice were fed the plant lipid daily from 7 days after EAE induction to assess the effects on acute disease and from day 25 to assess the effects on disease relapse. The clinical incidence and histological manifestations of acute EAE, and the clinical relapse phase of chronic relapsing EAE (CREAE) were markedly inhibited by omega-6 fatty acid feeding. A significant increase in the production of TGF-beta1 in response to concanavalin A (Con A) at day 13 and a significant increase in TGF-beta1 and PGE2 to Con A, PPD and MOG peptide (92-106) at day 21 were detected in spleen mononuclear cells from fatty acid-fed mice. There was no difference in interferon-gamma, IL-4 and IL-2 production between the fatty acid-fed and control groups. Significantly higher TGF-beta mRNA expression was found in the spleens of omega-6 fatty acid-fed mice at day 21. There were no differences in spleen cell proliferative response to Con A, PPD and MOG peptide (92-106). Biochemical analysis of spleen cell membrane fatty acids revealed significant increases in the eicosanoid precursor fatty acids dihomo-gamma-linolenic acid and arachidonic acid in response to gamma-linolenic acid feeding, indicating rapid metabolism to longer chain omega-6 fatty acids. These results show that oral feeding of gamma-linolenic acid-rich plant lipid markedly affects the disease course of acute EAE and CREAE and is associated with an increase in cell membrane long chain omega-6 fatty acids, production of PGE2 and gene transcription and, on activation, secretion of TGF-beta1.

Blood polymorphonuclear leukocytes from the majority of sickle cell patients in the crisis phase of the disease show enhanced adhesion to vascular endothelium and increased expression of CD64.

There is increasing interest in the role of blood polymorphonuclear leukocytes (PMNs) in the pathogenesis of sickle cell crisis. We studied the adherence of PMNs from 18 sickle cell patients in crisis, 25 out of crisis, and 43 healthy subjects (controls) to monolayers of human umbilical cord endothelium that were either untreated or pretreated with tumor necrosis factor alpha (TNFalpha). Overall, the PMNs from patients in crisis were more adherent than control PMNs to untreated endothelial monolayers (mean 53% increase; P < .001) and TNFalpha-treated monolayers (mean 41% increase; P < .002). Increased adhesiveness was not associated with an abnormal expression of CD11a, CD11b, CD11c, CD18, CD62L, or CD15. There was an increase in the number of PMNs expressing CD64 in patients in crisis (median value, 44%) compared with patients out of crisis (median, 21%; P = .025) and controls (median, 6.5%; P < .001). Sera from patients in crisis had normal levels of granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, interferon-gamma, TNFalpha, interleukin-1 (IL-1), IL-6, or IL-8 and did not modify the adherence of PMNs or their expression of CD64. Only IFN-gamma induced CD64 expression on PMNs, but this effect was not associated with enhanced binding to endothelium. Because PMNs bound to endothelial monolayers were CD64(+) and CD64-enriched PMNs were 7 times more adherent to endothelial monolayers than CD64-depleted PMNs, it is likely that CD64 is a marker of adherent PMNs. Two of the three anti-CD64 antibodies used in our antibody blocking studies (clones 32.2 and 197) partially inhibited the binding of sickle cell PMNs to untreated endothelium (mean inhibitions of 33% [P = .01] and 21% [P = .03], respectively), whereas only one (clone 197) inhibited binding to TNFalpha-treated endothelium (mean inhibition, 29%; P = . 004). In some patients with sickle cell disease, an enhanced PMN adhesion to vascular endothelium could contribute to the vascular occlusion that characterizes the acute crisis of the disease.

Human antiretinal antibodies in toxoplasma retinochoroiditis.

BACKGROUND/AIMS: Toxoplasma retinochoroiditis (TR) is an important cause of blindness and visual morbidity, affecting young adults. It has been postulated that some of the retinal damage observed in TR is due to antiretinal autoimmune mechanisms. METHODS: Humoral antiretinal autoimmunity in TR was investigated by indirect immunofluorescence (IIF) on normal human cadaveric retina and by a human retinal S-antigen ELISA. 36 patients with TR were separated on clinical grounds into those with first recurrence of disease (n = 18) or those with multiple recurrences (n = 18). Patients were also segregated into those with active (n = 28) or quiescent disease (n = 8). Serum from 16 normal controls (six with positive toxoplasma serology and 10 without) with no evidence of eye disease and 12 patients with idiopathic retinal vasculitis (IRV) were also tested. RESULTS: Sera from 34 of the 36 patients (94%) with TR demonstrated photoreceptor layer reactivity by IIF contrasting with six of 16 normal controls (p = < 0.001) and three of 12 IRV patients (p = < 0.001). Titres of antiphotoreceptor antibody were also higher among TR patients than controls. Sera from 27 of the 36 TR patients, 10 of 16 normals, and nine of 12 retinal vasculitis patients possessed anti-human retinal S-antigen antibodies at a titre of 1:400 or more as assessed by ELISA (p = > 0.05). Antiretinal autoantibody as detected by IIF did not run in parallel with S-antigen reactivity. CONCLUSIONS: The data indicate that the extent of antiretinal reactivity within TR is not accounted for by anti-S-antigen antibodies alone. This remarkably high prevalence of antiphotoreceptor antibody in TR as opposed to that found in either healthy or disease controls suggest that these antibodies may be co-pathogenic in toxoplasma retinochoroiditis.

A new method for studying the selective adherence of blood lymphocytes to the microvasculature of human retina.

PURPOSE: To develop a sensitive and reproducible technique for measuring the adherence of blood lymphocytes to vessel walls exposed in sections of human retina and for examining the role of lymphocyte and vascular adhesion molecules in these events. METHODS: Cryostat sections of human retina were overlaid with blood lymphocytes from healthy subjects, and experimental conditions were sought by which preferential attachment of the cells occurred to blood vessel walls in the retinal sections. Adherent lymphocytes were identified by staining with methyl green-thionine, and transected blood vessels were identified by their structure and by staining of basement membranes with periodic acid-Schiff. The adherence of enriched preparations of CD4+ (T-helper) and CD8+ (T-cytotoxic) lymphocytes, of interleukin-2 (IL-2)-activated cells, and of lymphocytes from patients with ocular Behçet's disease was examined. The distribution of adhesion molecules on retinal vessel walls was determined by immunohistochemistry, and the contribution of leukocyte integrins to lymphocyte binding was studied by blocking experiments with monoclonal antibodies. RESULTS: The optimal selectivity of blood lymphocyte attachment to retinal vessel walls occurred when purified lymphocytes were suspended in culture medium with 10% fetal calf serum and overlaid onto retinal sections for 30 minutes at 23 degrees C with gentle agitation. Under these conditions, 92% of the lymphocytes that adhered to the section were confined to the retinal microvasculature, and CD4+ T cells were more adherent than CD8+ T cells (P < 0.01). Prior exposure of normal lymphocytes to IL-2 enhanced their binding to retinal blood vessels, and lymphocytes from patients with Behçet's disease showed supranormal vascular adherence (P < 0.005). Many transected vessels stained positively for CD31; PECAM (mean 62%), CD54; ICAM-1 (mean 73%), CD62E; E-selectin (mean 35%), CD62P; P-selectin (mean 61%), and CD106; VCAM-1 (mean 42%). However, these vascular adhesion molecules occupied < 20% of the area of the blood vessel walls. Lymphocyte adhesion to the retinal vessels was more dependent on CD29 (the common chain of the beta 1 integrins) expression than either CD11a/CD18 or CD49d. CONCLUSIONS: This technique allows measurements to be made of lymphocyte adherence to vascular and nonvascular structures of retina ex vivo. Extension of this approach to the study of leukocyte adherence to sections of pathologic retina may be of clinical and experimental applicability in understanding mechanisms of retinal inflammation.

Lymphocyte-endothelial cell interactions in multiple sclerosis: disease specificity and relationship to circulating tumour necrosis factor-alpha and soluble adhesion molecules.

This study addressed two questions; first, whether the supranormal adherence of blood lymphocytes from patients with multiple sclerosis (MS) to endothelial cell monolayers treated with tumour necrosis factor-alpha (TNF alpha) was a feature common to other inflammatory disorders; and second, whether the adherence properties of blood lymphocytes from MS patients were related to changes in disease activity and to levels of circulating TNF alpha and soluble adhesion molecules. In the first part of the investigation, lymphocytes from 14 patients with MS were more adherent to TNF alpha-treated endothelial cells (P < 0.01) than those from healthy controls, whereas the adherence properties of lymphocytes from 12 patients with rheumatoid arthritis, eight patients with psoriasis and ten patients with neurological diseases other than MS were normal. In the second phase of the work, measurement of the adhesive properties of lymphocytes isolated at monthly intervals from a further six MS patients over a 5-8 month period, found that changes in binding to TNF alpha-treated endothelial cells, directly paralleled changes in circulating levels of TNF alpha (r = 0.77; P < 0.001) and soluble vascular cell adhesion molecule-I (sVCAM-1) r = 0.67; P = 0.001). An increase in disease activity, measured by T2-weighted and gadolinium-enhanced magnetic resonance imaging of the central nervous system (CNS), occurred in two patients and was associated with heightened lymphocyte adhesiveness and a rise in serum TNF alpha levels. Further analysis of the 34 serum samples from the six MS patients revealed a direct relationship between the concentration of sL-selectin and soluble intercellular adhesion molecule-I (sICAM-I) (r = 0.65; P < 0.001) and between sL-selectin and sTNF alpha (r = 0.42; P < 0.02). These findings support the view that disease activity in MS is associated with an increased adhesive interaction of blood lymphocytes with vascular endothelium at inflammatory sites within the CNS.

Intercellular adhesion molecule-1 in proliferative vitreoretinopathy.

PURPOSE: To measure vitreous levels of the soluble intercellular adhesion molecule (sICAM-1) in eyes with rhegmatogenous retinal detachment (RRD) complicated or uncomplicated by proliferative vitreoretinopathy (PVR) to investigate whether levels of this molecule related to history of previous retinal surgery or to the duration and severity of PVR. METHODS: The authors measured vitreous sICAM-1 by enzyme-linked immunosorbent assay in 28 eyes with PVR and 35 eyes with uncomplicated RRD. Vitreous from 10 eyes with macular holes and from 12 cadaveric eye donors were used as control specimens. RESULTS: Vitreous sICAM-1 levels were higher in the group with RRD complicated by PVR as a whole than in the group with RRD alone or in the control groups. In patients with no previous retinal surgery, there was no difference in vitreous sICAM-1 levels between the groups with RRD alone and RRD complicated by PVR. However, in patients who had undergone previous external surgery, those with PVR showed higher levels of vitreous sICAM-1 than those with RRD alone. In PVR, raised levels of sICAM-1 were associated preferentially with a history of previous vitrectomy as well as with a longer duration of the condition, although these levels were not related to the grade of PVR. In eyes with RRD alone, the levels of sICAM-1 were not enhanced with the duration of the detachment. Despite showing high vitreous levels of sICAM-1, patients with PVR did not exhibit increased serum levels of this adhesion molecule. CONCLUSIONS: The current observations suggest that those persons in whom PVR develops may have an impairment of the mechanisms that control the inflammatory response to retinal trauma. Persistently raised vitreous levels of sICAM-1 point to the continued operation of cytokine-mediated vascular reactions at the blood-retinal barrier.

Human blood dendritic cells: binding to vascular endothelium and expression of adhesion molecules.

To investigate the binding properties of dendritic cells (DC) to vascular endothelium, a comparative analysis was undertaken of DC, monocytes and lymphocytes isolated from the blood of 25 healthy subjects using monolayers of human umbilical vein endothelial cells as the adherence substrate. More blood DC (mean 24% adherence) were adherent to endothelial monolayers than monocytes (mean 18%; P < 0.001) and lymphocytes (mean 12%; P < 0.001). When the monolayers were pretreated with tumour necrosis factor-alpha (TNF-alpha) all leucocyte populations exhibited an increased attachment, but there was still greater binding of DC (mean 37% adherence) in comparison with monocytes (mean 23%; P < 0.001) and lymphocytes (mean 18%; P < 0.001). Flow cytometric analysis revealed that in relation to monocytes and lymphocytes the DC had a higher surface expression of the adhesion molecules CD11a (P < 0.05), CD11c (P < 0.005) and CD54 (P < 0.005) but a lower prevalence of cells bearing CD49d (mean 38%; P < 0.05) and the homing receptor CD62L (mean 14%; P < 0.001). CD1a was present on 22% of DC and virtually absent from the surface of monocytes and lymphocytes. The intensity of expression of the beta1-integrins, CD49c, CD49d and CD49e was greater on DC than lymphocytes and monocytes (P < 0.05). Antibody blocking studies demonstrated that DC binding to untreated and TNF-alpha-treated endothelium was dependent upon the expression of CD11a, CD18 and CD49d, and the simultaneous application of anti-CD18 and anti-CD49d antibodies produced an approximate 70% inhibition of adhesion (P < 0.001). Thus, the expression of both beta1- and beta2-integrins contributes to the adhesive interaction between DC and endothelium.

Detection and characterisation of anti-endomysial antibody in coeliac disease using human umbilical cord.

OBJECTIVES: To verify the effectiveness of human umbilical cord (HUC) in the detection of anti-endomysial antibodies (AEA) in coeliac disease and to characterize further these antibodies by studying tissue adsorption characteristics and antibody inhibition studies. METHODS: AEA were detected on HUC and primate oesophagus in a blind study, using sera from 46 patients with untreated coeliac disease and 108 controls. Tissue adsorption studies were performed using homogenized tissue from rodent liver, HUC, primate oesophagus and human liver. Sera were adsorbed with each of these homogenates and antibody was detected using HUC, primate oesophagus and rat kidney. In the inhibition experiments AEA was detected on HUC, and inhibition of binding was attempted by pre-incubating the sections with antibodies against collagen types I, III and IV. RESULTS: The sensitivity of AEA was 91% when detected on HUC, 89% when detected on primate oesophagus (93% and 91%, respectively, after exclusion of 1 patient with IgA deficiency). Specificity was 100% for both assays. Tissue adsorption studies showed identical results for AEA detected on both HUC or primate oesophagus, whereas antireticulin antibody was adsorbed only by rodent tissue. Blocking of the HUC with anticollagen antibodies did not prevent binding of AEA. CONCLUSIONS: HUC is an effective substrate for the detection of AEA and may be superior to primate oesophagus. The antibody detected by HUC shows identical tissue adsorption specificities to that detected on primate oesophagus.

Binucleated and multinucleated forms of plasma cells in synovia from patients with rheumatoid arthritis.

A morphological examination of synovial tissue from 25 patients with rheumatoid arthritis revealed that binucleated or multinucleated plasma cells were present in all samples and absent in synovia obtained from 16 control patients. Plasma cells containing two, three of four nuclei constitute a mean 3% of the total plasma cell population. They were always found amongst plasma cell infiltrates and in close association with small blood vessels. Ultrastructural analysis found no evidence of cellular membranes separating the individual nuclei in binucleated or multinucleated plasma cells, suggesting that the cells did not arise from fusion. Some of these plasma cells had a diameter approaching 100 microns, and many were in intimate contact with macrophages. The demonstration of a few cells with mitotic figures within the infiltrates suggests that the maintenance of plasma cell numbers in rheumatoid synovium may depend, in part, upon their local proliferation.

Adhesion molecules are upregulated on dendritic cells isolated from human blood.

This study investigated whether the high expression of adhesion molecules on enriched preparations of circulating dendritic cells (DCs) was an intrinsic property of the cells or whether it was a consequence of the procedure used to isolate them from blood. Expression of the beta 1, beta 2 integrins (CD11/CD18 family) and other adhesion molecules on DCs in whole blood was compared with that on isolated DCs. Dendritic cells were identified by flow cytometry as leucocytes that were positive for human leucocyte antigen (HLA)-DR, but negative for CD3, CD14, CD16, CD19 and CD56. In contrast to a minority of DCs in whole blood, the majority of isolated DCs expressed the beta 2 integrins and there were a greater number of cells bearing CD44, CD54 and some of the beta 1 integrins (notably CD49b, CD49d, CD49e and CD29). An increase in the proportion of DCs bearing adhesion molecules was generally apparent at the isolation stage when mononuclear cells, which had been incubated overnight, were centrifuged on a metrizamide gradient to enrich for cells of low density. Inclusion of an inhibitor of protein glycosylation and exocytosis (brefeldin A) at all stages of separation partially prevented an increase in the percentage of DCs bearing CD18, C29 and C54 whereas the inclusion of cycloheximide (an inhibitor of polypeptide synthesis) interfered with increases in the percentage of cells bearing CD29 and CD54. Neither of these antagonists had an effect on the intensity of adhesion molecule expression. We suggest that some of the adhesion-dependent functions of isolated DCs are caused, in part, by an upregulation of surface adhesion molecules induced by the enrichment procedure.

IL-6 acts on endothelial cells to preferentially increase their adherence for lymphocytes.

Using a quantitative monolayer adhesion assay, the current report shows that treatment of human umbilical vein endothelial cells (HUVEC) with IL-6 increases their adhesiveness for blood lymphocytes, particularly CD4+ cells, but not for polymorphonuclear cells and monocytes. This effect, which was most pronounced when using low concentrations of the cytokine (0.1-1.0 U/ml) and a short incubation period (4h), was also apparent with microvascular endothelial cells and a hybrid endothelial cell line. Skin lesions from patients with mycosis fungoides contain high levels of IL-6, and blood lymphocytes from patients with this disorder also exhibited an enhanced adhesion to IL-6-treated HUVEC. The cytokine enhanced intercellular adhesion molecule-1 (ICAM-1) expression and induced the expression of vascular cell adhesion molecule-1 (VCAM-1) and E-selectin on endothelial cells. Antibody blocking studies demonstrated that the vascular adhesion molecules ICAM-1, VCAM-1 and E-selectin and the leucocyte integrin LFA-1 all contributed to lymphocyte binding to endothelium activated by IL-6. It is proposed that IL-6 may be involved in the recruitment of lymphocytes into non-lymphoid tissue.

Enhanced binding of lymphocytes from patients with multiple sclerosis to tumour necrosis factor-alpha (TNF-alpha)-treated endothelial monolayers: associations with clinical relapse and adhesion molecule expression.

This study investigated the adherent properties and adhesion molecule expression of blood mononuclear cells (MNC) from a total of 84 patients with multiple sclerosis (MS). The MNC from MS patients were significantly more adherent than cells from normal healthy subjects to endothelial monolayers pretreated with 0.01 U/ml TNF-alpha (103% increase; P = 0.002), 0.1 U/ml TNF-alpha (80% increase; P < 0.01) and 1.0 U/ml TNF-alpha (41% increase; P < 0.02), and to endothelium pretreated with 10 U/ml IL-1 beta (44% increase; P < 0.05) and 100 U/ml interferon-gamma (IFN-gamma) (100% increase; P < 0.05). This augmented adhesion was a property of the lymphocytes, in particular CD4+ cells, and was inversely related to the time of onset of clinical relapse. The percentage of lymphocytes bearing the adhesion molecules CD49d, CD29 and CD62L was increased in MS blood, but the level of CD29 and CD62L expression was reduced. We infer that circulating lymphocytes in MS are predisposed to cross endothelial barriers at sites where inflammation has already commenced.

Evidence of chronic inflammation in retina excised after relaxing retinotomy for anterior proliferative vitreoretinopathy.

BACKGROUND: Epiretinal membranes from eyes with proliferative vitreoretinopathy (PVR) frequently express molecules associated with chronic inflammation. To investigate the extent to which inflammation may compromise the detached retina, we determined the expression of inflammatory molecules in anterior retina removed after relaxing retinotomy for retinal detachment complicated by anterior PVR. METHODS: Surgical retinal specimens were studied immunohistochemically for the distribution of the vascular cell adhesion molecules VCAM, E-selectin, P-selectin, ICAM and PECAM and for the presence of the cytokine TNF alpha and of T lymphocytes (CD3-positive cells), macrophages (CD68-positive cells) and HLA-DR molecules. The findings were compared with those in control cadaveric retina. RESULTS: Aberrant expression of ICAM-1 was observed in four of nine retinal specimens from eyes with PVR, whereas its expression in control retinas was confined to the external limiting membrane and ganglion cell layers. PECAM was observed in seven of nine surgical retinal specimens and in four of five controls. E-selectin and P-selectin were expressed within the luminal aspects of four of nine retinal specimens from eyes with PVR, and VCAM was present in three of nine surgical specimens investigated. All cadaveric control retinas were negative for E-selectin and VCAM, whilst one was positive for P-selectin. Staining for TNF alpha was observed within luminal aspects and walls of retinal vessels from eight of nine surgical specimens, but was not seen in any of the cadaveric controls. T lymphocytes and cells expressing the macrophage marker CD68 were identified in two and seven of nine diseased retinas respectively, but not in any of the controls. Cells staining for HLA-DR were observed in eight of nine surgical retinal specimens and in three of five controls. CONCLUSION: The present findings indicate that retina from eyes with advanced PVR may itself be subject to inflammatory changes, and indicate that the PVR process is not limited to retinal membranes, but involves a more widespread distribution of inflammation than is generally appreciated.

Distribution of TNF alpha and its reactive vascular adhesion molecules in fibrovascular membranes of proliferative diabetic retinopathy.

AIMS: This study investigated the presence of the cytokine tumour necrosis factor alpha (TNF alpha) and the vascular adhesion glycoproteins ICAM-1, VCAM-1, E-selectin, P-selectin, and PECAM within fibrovascular membranes of eyes with proliferative diabetic retinopathy (PDR). METHODS: The presence of these molecules was determined by immunohistochemical staining using monoclonal antibodies and the APAAP technique. RESULTS: Staining for TNF alpha was observed on the retinal vascular endothelium of five of 12 specimens, on infiltrating cells within all membranes, and on the extracellular matrix of nine specimens. This staining wa abolished by absorption of the monoclonal antibody with human recombinant TNF alpha. Likewise, ICAM-1 staining was given by infiltrating cells and extracellular matrix of nine membranes and by the endothelium of three of the specimens. VCAM-1, E-selectin, and P-selectin staining was observed on the vascular endothelium of 5/12, 4/12, and 3/12 epiretinal membranes respectively. PECAM was expressed by the endothelium of 4/12 specimens, by infiltrating cells of 8/12 membranes, and also by the extracellular matrix of two of the specimens. CONCLUSION: The widespread distribution of TNF alpha and the nature of the adhesion molecules expressed by vascular endothelial cells in PDR membranes suggest that local activation of TNF alpha and enhanced expression of vascular cell adhesion molecules may play an important role in the development of the proliferative phase of diabetic retinopathy.

Blood monocytes in rheumatoid arthritis are highly adherent to cultured endothelium.

Monocytes from 17 patients with rheumatoid arthritis (RA) were more adherent than monocytes from 17 control patients to monolayers of pig aortic endothelium irrespective of whether sera was included (median 27-34% increase; p = 0.002) or omitted (median 27% increase; p = 0.022) from the culture media. When human umbilical vein endothelial cells were used as the adherence substrate, rheumatoid monocytes from an additional 21 patients demonstrated a median 31% (p = 0.004) and 20% increase (p = 0.004) in adhesion when compared with monocytes from 21 normal healthy subjects in the absence and presence of autologous sera, respectively. Activation of control monocytes with muramyl dipeptide or treatment with RA sera increased their attachment to endothelium (mean 34 +/- 14% increase; p < 0.001). The expression of the adhesion molecules CD11b (p < 0.005), CD18 (p < 0.005), CD62L (p = 0.01) was enhanced on rheumatoid monocytes, but antibody-blocking studies suggested that CD18 and CD62L were not responsible for the augmented binding of the rheumatoid cells. A subpopulation of rheumatoid monocytes possessed a very low net negative surface charge, a property that favours binding to vessel walls. We propose that many rheumatoid monocytes are predisposed for sheer-resistant adhesion to vascular endothelium.

The distribution and abnormal morphology of plasma cells in rheumatoid synovium.

This study assessed the distribution and structural features of plasma cells in rheumatoid synovial tissue. Plasma cells were found to be the predominant infiltrating mononuclear cells (mean 40%) in relation to lymphocytes and monocytes, and there was a direct relationship between their number in the infiltrates and the total number of mononuclear leucocytes (P = 0.007). Plasma cells were also seen in intimate contact with macrophages intermixed with synovial lining cells, and closely associated with small blood vessels. They often surrounded these blood vessels and sometimes were seen lying within the vessel walls themselves. Ultrastructural analysis revealed that many synovial plasma cells were considerably larger than plasma cells of a normal size and possessed a marked distension of the cisternae of rough endoplasmic reticulum. Furthermore, plasma cells in close proximity to blood vessels often appeared to be undergoing migration. These observations imply that in rheumatoid synovium, plasma cells are metabolically very active and occupy a pivotal position for the secretion of antibodies into both the vascular and the extravascular compartments.

Selective binding of peripheral blood lymphocytes to the walls of cerebral vessels in frozen sections of human brain.

In order to identify the factors that control the binding of blood leucocytes to cerebral blood vessels we have modified and applied the frozen section assay of Stamper and Woodruff to the study of human brain. Cryostat sections of brain tissue obtained at post mortem were overlaid with blood lymphocytes and experimental conditions were defined which permitted optimum binding of the cells to transected blood vessel walls. The maximal binding of lymphocytes to cerebral vessels occurred when 6 x 10(6) lymphocytes were overlaid onto brain sections for 30 min at 7 degrees C with gentle agitation. Only a small proportion (0.01%) of the added lymphocytes bound to exposed cerebral vessels. However, lymphocytes were far more adherent than monocytes and polymorphonuclear cells (7-fold and 11-fold respectively: p < 0.001) and activation of lymphocytes with IL-2 enhanced their binding to blood vessel walls (mean 130% increase; p < 0.03). Further analysis revealed that CD4-positive T lymphocytes were the predominant cell population binding to the blood vessels. Antibody blocking studies showed that lymphocyte binding to cerebral blood vessels was inhibited by pretreating the lymphocytes with anti-CD11a, anti-CD18 or anti-CD49d (p < or = 0.02) and immunohistochemical studies revealed the presence of the counter-receptors ICAM-1 (CD54) and VCAM-1 (CD106) for these adhesion molecules in addition to the presence of E-selectin (CD62E) and P-selectin (CD62P) on the cerebral blood vessels. The establishment of a technique in situ which measures selective binding of CD4-positive peripheral lymphocytes to sections of cerebral blood vessels will assist in the molecular characterization of factors that control the interaction of leucocytes with the blood-brain barrier in health and disease.