Francesca Vargas

Evidence of expression of endotoxin receptors CD14, toll‐like receptors TLR4 and TLR2 and associated molecule MD‐2 and of sensitivity to endotoxin (LPS) in islet beta cells

CD14, a GPI‐linked membrane protein, is a component of the lipopolysaccharide (LPS) receptor complex, one of the pattern‐recognizing receptors (PRR) expressed by myeloid lineage cells. Here we report that CD14, the functionally linked toll‐like receptor molecules, TLR2 and TLR4, and the associated molecule MD‐2 are expressed in endocrine cells of the human pancreatic islets. CD14 expression in human pancreatic islets was determined by immunofluorescence staining of tissue sections and primary cultures, and confirmed by flow cytometry of dispersed normal islets and SV40‐transformed islet cells (HP62). The latter cells synthesized and secreted CD14 in response to lipopolysaccharide (LPS) in a time‐ and dose‐dependent manner. Reverse transcription polymerase chain reaction (RT‐PCR)‐Southern was positive for CD14, TLR2, TLR4 and MD‐2 in human pancreas, purified islets and HP62 cells. In vitro experiments using rat islets (also positive for CD14 by RT‐PCR) and HP62 cells showed that LPS regulates glucose‐dependent insulin secretion and induces inflammatory cytokines [interleukin (IL)‐1α, IL‐6 and tumour necrosis factor (TNF)‐α]. The functional expression of CD14 and associated molecules in islet β cells adds a new pathway that islet cells may follow to adjust their function to endotoxaemia situations and become vulnerable to the inflammatory events that occur during diabetogenic insulitis.

Endotoxin contamination may be responsible for the unexplained failure of human pancreatic islet transplantation.

Clinical transplantation of human islets has a disappointingly low rate of success. We report here the identification of a possible causative factor: endotoxin present in the collagenase preparations used to disperse the pancreatic tissue before islet purification and transplantation. Supporting evidence includes (1) detection of unexpectedly high levels of endotoxin in most collagenase solutions currently used to digest human pancreases; (2) demonstration that supernatants generated during islet separation are able to induce the inflammatory cytokines interleukin (IL)-1, IL-6, and tumor necrosis factor-alpha (TNF-alpha) in macrophages; and (3) induction of IL-1, IL-6, and TNF-alpha in the islets during the separation procedure. Cytokine expression was assessed by reverse transcription-polymerase chain reaction and, for TNF-alpha, confirmed by enzyme-linked immunoabsorbent assay. It is proposed that endotoxin and locally induced cytokines carried over with the graft activate the endothelium and promote lymphomonocytic infiltration of grafted islets and surrounding liver tissue favoring primary nonfunction and early rejection. These results also have implications for the numerous experimental procedures that use collagenase, and they point to possible ways to improve islet preparation and transplantation protocols.

Characterization of neural cell adhesion molecule (NCAM) expression in thyroid follicular cells : induction by cytokines and over-expression in autoimmune glands

NCAM (CD56) is a cell surface glycoprotein of the immunoglobulin superfamily expressed on neuroendocrine and natural killer (NK) cells which has considerable molecular heterogeneity due to differential splicing and post‐translational modifications. NCAM has been detected in the thyroid follicular cells (thyrocytes) immunohistological. We report here the molecular form, the modulation by cytokines and the levels of expression in thyroid pathology. By using a panel of MoAbs to NCAM on Western blots from thyrocyte extract we have determined that these cells express the 140‐ and 180‐kD forms of NCAM, Exposure of primary cultures of thyrocytes to interferon‐gamma (IFN‐Γ), and even more, to the combination of IFN‐Γ plus tumour necrosis factor‐alpha (TNF‐α) induced a clear increase in the expression of NCAM as assessed by FACS analysis. NCAM expression in thyrocytes was assessed by immunofluorescence in 59 surgical specimens of thyroid glands, and was found increased in 11/17 (64%) of Graves’, in 5/25 (20%) of multinodular goitre (MNG) and in occasional adenoma glands. No correlation was found with the expression of HLA class I. class II or the degree of lymphocytic infiltration scored in adjacent sections, but it was often seen in areas infiltrated by macrophages. In conclusion, NCAM is an adhesion molecule whose expression is clearly increased in thyrocytes in autoimmune glands, probably as a consequence of exposure to cytokines locally released. Since one of the forms of NCAM expressed by thyrocytes has the capability to generate intracellular signal it may play a role in normal thyroid function. In addition, NCAM may facilitate the recognition of thyrocytes by lymphocytes, particularly by NK CD56+ lymphocytes.

γσ Lymphocytes in endocrine autoimmunity: evidence of expansion in Graves' disease but not in type 1 diabetes

Endocrine autoimmune disorders are mediated by T cell‐dependent responses to organ‐specific antigens, but the mechanisms initiating the process remain unknown. Lymphocytes whieh use the γδ heterodimer as T ceii receptor (TCR) for antigen constitute a distinct subset of T cells whose function remains elusive. In order to investigate their possible involvement in endocrine auloimmunity we have determined the proportion of γδ T cells in the peripheral biood of 23 patients with type 1 (insulin‐dependent) diabetes mellitus (type‐1 DM) and 30 patients with autoimmune thyrotoxicosis (Graves’ disease). T lymphocyte TCR expression was assessed by fluorescence‐activated flow eytometry on peripheral blood mononuclear cells using MoAbs UCHTI (CD3), TCR 51 (γδ TCR), WT31 and βF1 (αβ TCR) and both the percentage of T cells expressing γδ and the ratio γδ/αβ were calculated. In the diabetie patients γδ cells were not significantly different from (he control group (7.7 ± 54%versus 8.0 ± 5.5%) of T eells, P NS). There was no relation between the proportion of γδ lymphoeytes and the presence ol’ islet cell antibodies (ICA) in the sera. The Graves’ patients showed a tendency towards a higher proportion of γδ T lymphocytes than the controls (γδ/αβ ratios: 0.095 ± 0.047 versus 0.063 ± 0.022, P= 0 03). In 14 Graves’ patients the number of γδ were measured in paired samples of peripheral and inlrathyroidal lymphocytes, demonstrating an expansion of γδ within the thyroid glands (0.21 ± 0.3 versus 0.095 ± 0.047, P= 0.032). Immunohistochemical studies showed that γδ celts were scattered among the predominant αβ lymphoeytes infiltrating the thyroid gland and that they aeeount for 10% of intraepitheliai lymphocytes. No relation was found between the increase of γδ lymphocytes and any clinieai features.

Expression of Transporter Associated With Antigen Processing–1 in the Endocrine Cells of Human Pancreatic Islets: Effect of Cytokines and Evidence of Hyperexpression in IDDM

A possible role of transporter associated with antigen processing (TAP)-l in the pathogenesis of IDDM has been investigated by examining the level of TAP-1 expression in the islets of IDDM pancreas and by studying in vitro the effect of interferon (IFN)-γ, IFN-α, and tumor necrosis factor-α in TAP-1 expression by cultured islet cells. A remarkable hyperexpression of TAP-1 has been found in the endocrine cells (β and non-β) of IDDM islets, which constitutes first evidence of hyperexpression of this molecule in the target organ of an autoimmune disease. TAP-1 hyperexpression correlated clearly with HLA class I hyperexpression but only very partially with HLA class II ectopic expression. IFN-γ and IFN-α, both cytokines putatively implicated in IDDM pathogenesis, were capable of inducing TAP-1 protein (as assessed by immunofluorescence flow cytometry) and message (by Northern blot analysis and reverse transcription polymerase chain reaction). These findings suggest that under the influence of cytokines (most probably IFN-α) β-cells may express in their surface a high density of HLA class I–peptide complexes that may facilitate their recognition and lysis by low-affinity CD8+ T-cells.

Expression of glutamic acid decarboxylase (GAD) in the α, β and δ cells of normal and diabetic pancreas: implications for the pathogenesis of type I diabetes

One of the paradoxes of insulin‐dependent diabetes mellitus is that the destruction of the pancreatic islets’ endocrine cells is restricted to the insulin‐producing β cells, whereas the main autoantibodies, islet cell antibodies (ICA), are directed against all endocrine islet cells. GAD has recently been proposed as the main target of the humoral and cellular autoimmune attack to the islets, and since in rat pancreas this enzyme was expressed only in the β cells, this provided an explanation for the cell specificity of the destructive process. The finding of GAD‐positive cells in the islets of two diabetic patients, one of whom had completely lost the β cells, led us to study in detail thedistribution of GAD in normal human islet ceils using a panel of GAD aniiscra and the double indirect immunofluorescence technique on cryostat sections, monolayer cultures and cytosmears. The results showed that GAD is present not only in the cytoplasm of β cells but also in 69% of the α and 27% of the δ cells. GAD was not present, however, on the surface of the islet cells. These results suggest that the cellular distribution of GADcan not by itself explain the selectivity of β cell destruction in insulin‐dependent diabetes mellitus.

Endotoxin activity of collagenase and human islet transplantation

Vol 350 • August 30, 1997 641 which ranged from 10 to 26 times per day before treatment, decreased to 6–12 times per day (33%–58%), and this effect was detected as soon as the first day after treatment. Three patients were incontinent and became dry most days. Improvement was sustained up to 3 months, the longest follow-up available. A rise in maximum cystometric capacity (MCC) occurred in four of these patients, with maximum increment ranging from 76 mL to 596 mL (51% and 900% of pretreatment MCC, respectively). In a sixth patient, who had 18 mL of MCC and collected urine on pads, no clinical improvement occurred despite a continuous increase in MCC which reached 76 mL in one occasion (330% rise). The only patient (incontinent after transverse myelitis) who did not respond clinically or urodynamically to RTX had experienced a similar failure after capsaicin. We gave RTX (50 nmol/L) to two patients who have never received capsaicin. Both emptied their bladders by intermittent catheterisation but still leaked due to nonvoluntary bladder contractions. Again, discomfort evoked by RTX was minimum. In one patient, who was taking 5 mg oxybutynin twice daily without success, the addition of RTX brought continence on most days and increased MCC from 200 mL to 256 mL.

Advantages of using a cell separator and metrizamide gradients for human islet purification.

Human islet transplantation has a high rate of failure, often due to primary nonfunction, which suggests that islets are damaged during the processing of the pancreas. The preparation of human islets for transplantation is still a complex process that requires large teams of surgical and laboratory personnel. To overcome this problem, we have adopted the use of the IBM 2991 COBE cell separator and a metrizamide/Ficoll density medium that is easy to prepare. Twenty-seven pancreatic glands have been processed using the COBE cell separator, 23 of which were purified in metrizamide/Ficoll gradients and 4 in bovine serum albumin gradients. The results show an improvement of recovery and viability in these preparations when compared retrospectively with manual gradients. More importantly, the time required for purification was shortened to one fourth the usual time and total processing time is about half as long. Moreover, a team of two laboratory staff was regularly able to prepare islets for transplantation, reducing the separation time from 7 hr to 3.5 hr. We conclude that the automatic cell separator and metrizamide-based separation medium are useful modifications of current islet purification methods.