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Clin Exp Immunol. Aug 2003; 133(2): 208–218.
PMCID: PMC1808777

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

Abstract

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.

Keywords: CD14, diabetes, human β cells, LPS

INTRODUCTION

CD14 is a myeloid differentiation antigen described first in monocytes [1] but also expressed in neutrophils [2] and B lymphocytes [3]. CD14 is a 53–55 kDa membrane glycoprotein related to the newly described family of toll-like receptors (TLR) that also contains a leucine-rich repeat domain (as is the case in many TLRs). CD14 does not have a transmembrane domain and is anchored to the cell surface via a glycosyl–phosphatidyl–inositol (GPI) bond which confers mobility and easy secretion by shedding. The CD14 gene is located in chromosome 5 in humans in a region that contains other genes coding for myeloid cell receptors and growth factors [4]. CD14 is the receptor of the complex formed by lipopolysaccharide (LPS) with LBP (LPS binding protein), an acute phase response protein normally present in trace amounts in human serum [5]. Relatively high levels of secreted CD14 isoforms (sCD14) have been detected in the plasma, in culture supernatants from CD14-expressing cells [69] and in breast milk [10]. sCD14 modulates T cell [11] and B cell activation [12] and stimulates human endothelial cells [13]. CD14 requires the expression of co-receptor molecules for signal transduction. Some members of the TLR family (particularly TLR4) serve this essential function and may help to restrict the specificity of CD14 [14] and another molecule, MD-2, is required for enhancing signal transduction by TLR4 [15].

The idea that CD14 is a molecule expressed only in myeloid lineage cells has been challenged by the observation that CD14 is expressed in many cell types and tissues of mice injected previously with LPS, such as cardiac myocytes, kidney tubular cells, hepatocytes, bronchiolar and transitional epithelium. The function of CD14 in non-myeloid cells is still unknown [16].

During our studies aimed at characterizing the insulitis in the pancreas of a diabetic patient, we detected CD14 in the islets [17]. Given the crucial functional role of CD14 as a pattern recognition receptor (PRR) [11], we undertook the present study.

Here we provide evidence of CD14 expression in islet cells and this molecule appears to be a functional LPS receptor on β cells. TLR4 and TLR2 and the leucine repeat rich MD-2 proteins have also been detected in islet cells, thus suggesting that β cells express a functional LPS receptor.

MATERIALS AND METHODS

The protocols described in this report have been approved by the ethical committee of the Hospital Universitari Germans Trias i Pujol.

Substrates

Human pancreas and islet cell culture

 Tissue was obtained from 15 organ donors (nine men and six women, age range 12–60 years) whose pancreases were donated with the permission of the family, but not transplanted. Pancreas donors were tested for islet cell antibodies (ICA) to exclude prediabetic subjects. For immunofluorescence studies, blocks of tissue were snap-frozen and kept at − 80°C until used. Some of the glands were digested with Collagenase P (Roche, Basel, Switzerland) [18,19] and free islets were identified by staining with dithizone [20], dispersed with dispase (type II, 72 U/ml, Roche) and cultured on glass coverslips at 37°C in a 5% CO2/air humidified incubator as described [21]. The culture medium was 199 (Gibco, Paisley, UK) supplemented with 10% endotoxin-free FCS (Myoclone, Gibco) and antibiotics. Rat islets from adult Lewis rats (weighing 250–280 g) were prepared following a modified standard collagenase digestion method as described [19].

Cell lines

HP62 is an epithelial human cell line of insular origin generated by transfection of human islets with plasmid pX-8, containing the SV40 early region. This cell line retained insulin production during the first six passages and has been grown in continuous culture for 10 years without losing epithelial cell features [22]. NES2Y is an insular cell line generated from a nesidioblastosis pancreas that maintained proinsulin production during the first 30 passages [23]. RIN-5F is an islet cell rat cell line originally derived from an insulinoma [24].

The monocytic cell lines THP-1 [25] and MonoMac-6 [3] (DMS, Braunschweig, Germany) were used as positive controls. HT93, an SV40-transformed thyroid cell line [26], was used as negative control. As additional controls we used: Jurkat (T cell lymphoma); Nalm-6 (pre-B acute lymphoblastic leukaemia); and Daudi (Burkitt's lymphoma). Cells were maintained in RPMI-1640 with 10% endotoxin-free FCS (Myoclone, Gibco) and antibiotics as above. The Mono-Mac-6 cells culture medium was supplemented with insulin.

Indirect immunofluorescence staining protocols

Antibodies

The following antibodies were used: glutamic acid decarboxylase (GAD) c38 (rabbit anti-GAD 65/67, Dr Y. Wu, Kansas, KA, USA), guinea pig anti-insulin (ICN, Lislei, IL, USA), rabbit antiglucagon (Amersham) three MoAbs against CD14 (Cris-6, Dr R. Vilella, HCPB, Barcelona, Spain; MY4, Coulter, Hialeah, FL, USA and Leu M3, Becton-Dickinson, Mountain View, CA, USA), MoAb to CD68, a marker for macrophages (EMB11, Dako, Glostrup, Denmark) and MOPC-141 (as isotype-matched control, Sigma, St Louis, MO, USA).

Frozen sections

Consecutive cryostat sections, 5 µm, were air-dried and fixed in cold acetone. To block non-specific binding, 0·5% of calf serum or bovine serum albumin (BSA) was added to the phosphate buffered saline (PBS) used to dilute the antibodies. The sections were incubated sequentially with (i) MoAb to CD14; (ii) FITC-labelled goat antimouse IgG F (ab)2 serum; (iii) either guinea pig anti-insulin, rabbit antiglucagon or rabbit anti-GAD c38; and (iv) depending on the third layer, tetramethyl rhodamine isothiocyanate (TRITC) labelled goat antiguinea pig or goat antirabbit Ig (Southern, Birmingham, AL, USA) sera were used as fourth layer. Only sections containing more than 10 islets were evaluated under a UV microscope (Zeiss Axioplan, Wetzlar, Germany); the images were obtained with a high-resolution video camera, digitized and deconvolved (OpenLab 2·0, Improvision, Coventry, UK).

Viable islet cell monolayers were stained on the coverslips. The monolayers were incubated sequentially following a protocol similar to that described for tissue sections. A fixation step with formalin or methanol was added prior to insulin staining in order to permeabilize the membrane. The monolayers were examined under a UV microscope using × 40 and × 63 objectives. Three independent experiments were performed.

For flow cytometry analysis, aliquots of 105 islet cells were incubated sequentially at 4°C for 30 min with MoAbs to CD14 or to CD68 and FITC goat antimouse IgG F(ab)2-conjugated antiserum. The controls included unstained cells (autofluorescence control), cells stained with normal mouse serum at 1/500 and cells stained with MOPC-141 (as isotype-matched control) and followed by the conjugate (background control). In order to analyse cytoplasmic antigens, the cells were permeabilized (permeabilization Kit, Sera-lab, Crawley Down, Sussex, UK) prior to staining. Three independent experiments were performed. The number of CD14 molecules expressed on the human insular cells was estimated using calibrated beads (Qifikit, Biocytex, Marseille, France). The analysis was carried out in a FACScan cell analyser (Becton Dickinson).

Detection of cytokines and CD14 using RT-PCR

RNA was extracted from snap-frozen pellets as described [27]. cDNA was prepared by incubating 1 µg of denatured RNA with oligo-dT20 (final concentration: 1 µm) and Superscript™ RNase H-Reverse Transcriptase (Gibco/BRL, Gaithersburg, MD, USA), adding 2 U RNAsin (Promega, Madison, WI, USA) per sample. The product was heated to 95°C for 5 min and stored at − 70°C until used. PCR was performed by incubating 1·0 µl of the above cDNA with 10 mm Tris-Cl (pH 8·8), 50 mm KCl, 1·5 mm MgCl2, 0·1% Triton X-100, 0·2 mm of each dNTPs, 0·5 mm of each primer and 0·5 U Termus aquaticus DNA polymerase (Taq DNA pol, Gibco/BRL). The primers for interleukin (IL)-1α, IL-1β and tumour necrosis factor (TNF)-α were obtained from Stratagene (La Jolla, CA, USA). The primers and oligoprobes for CD14 (human and rat) and for TLR2, TLR4, DM2, GAPDH and β actin were designed (Oligo software from Molecular Biology Insights, Inc., Cascade, USA) always spanning at least one intron (Table 1). In order to normalize the cDNA samples, serial dilutions were amplified (24–30 cycles, below the amplification plateau) for GAPDH (human) or β actin (rat). RT-PCR products were electrophoresed in 2% agarose gels with ethidium bromide and visualized on an UV transilluminator. For Southern blotting, the gels were transferred to HybondTM-N nylon membranes (Amersham) in 0·4 N NaOH and UV cross-linked. The membranes were washed for 2 min in 2×SSC (1×SSC = 0·15 m NaCl/0·013 m sodium citrate) and prehybridized directly in 5×SSPE/10×Denhardt's solution/0·1% SDS/40 mg/ml salmon sperm DNA for 1 h before hybridization for 3 h at 47°C in the same solution plus 20 pmol of radiolabelled probe. The oligoprobe was labelled with γ(32P) dATP (Amersham) using T4 polynucleotide kinase (New England Biolabs, Beverly, MA, USA). The filters were washed under increasingly stringent conditions. Cronex X-ray film plates (DuPont) were exposed to the radiolabelled membranes at −70°C for 1–7 days. Three independent experiments were performed.

Table 1
List of primers used for the detection of CD14, toll-like receptors TLR4 and TLR2 and associated molecule MD-2 and PCR conditions

CD14 immunoprecipitation

THP-1 (1·5 × 106 cells/ml), HP62 and RIN cells (1 × 106 cells/ml), cultured in medium lacking l-methionine (Gibco) were labelled biosynthetically with TRAN35S-LABEL (13·46 mCi/ml, ICN, Irvine, CA, USA) for 18 h. Supernatants were filtered (0·22 µm filters) and desalted in Centricon-10 microconcentrators (Amicon, Beverly, MA, USA) with PBS containing 0·02% (w/v) sodium azide, 2 mg/ml l-methionine and lysis buffer (10 mm Tris-HCl, pH 7·4, 150 mm NaCl, 0·5% (w/v) NP40, 0·02% (w/v) sodium azide, 2 mmphenyl methyl sulfonil fluoride (PMSF). Supernatants were concentrated 10×. The cells were detached mechanically and washed in ice-cold PBS containing 0·02% (w/v) sodium azide and the cell lysates were prepared immediately.

The labelled cells were lysed and the insoluble material was removed by centrifugation at 4°C. The cell lysates were precleared with MOPC-141, an irrelevant MoAb. The precleared material was incubated with MY4 (MoAb to CD14) overnight at 4°C and treated with protein G-Sepharose beads precoated with goat antimouse sera. Immunoprecipitates were washed with ice-cold lysis buffer and resuspended in 80 µl of reducing sample buffer [1·6% (w/v) SDS, 0·125 m Tris, 12% glycerol, 4% 2-mercaptoethanol, 0·03% (w/v) EDTA]. Boiled samples were run on 10% acrylamide gels (Bio-Rad, Richmond, CA, USA). Fluorographic treatment of the gels containing 35S-labelled proteins was performed (Amplify, Amersham), and the dry gels were exposed for 2–14 days. Molecular weights were determined by comparing the middle of the sample band migration to the migration of m.w. standards (14C methylated proteins, 220 kDa-14·3 kDa, Amersham). sCD14 was measured in HP62 culture supernatant at different time-points and LPS concentrations by ELISA (IBL, Hamburg, Germany), lower detection limit of 0·5 ng/ml. Two independent experiments were performed.

Functional studies

Glucose-dependent insulin release

In the first series of experiments (short-term effects), insulin release and content were measured after static incubation for 90 min. Groups of eight hand-picked rat islets were incubated in 1 ml of bicarbonate-buffered medium with BSA (5 mg/ml), d-glucose (2·8–16·7 mm) and LPS (0–100 ng/ml). The supernatants were removed and the islets were sonicated in 500 µl of acid-alcohol solution. In a second series of experiments (long-term effects), groups of 150–200 islets were cultured for 18 h in 5·5 mm d-glucose and either 0, 1 or 100 ng/ml LPS. After incubating for 18 h, insulin secretion was measured as described above for 90 min. In order to determine whether the effect induced by LPS was reversible, the islets were cultured in LPS-containing medium for 18 h, washed and incubated for 18 h before measuring insulin release and content. Four different independent experiments with triplicate cultures were performed.

RIA for insulin (CIS Biointernational, Gif-Sur-Yvette, France; detection threshold, 2·5 µU/ml; coefficient of variation 6% intra-assay and 8% interassay) had a lower detection limit of 2·5 µU/ml and a coefficient of variation within and between assays of 6% and 8%, respectively. Insulin release is expressed as µU insulin/islet per 90 min. Results are expressed as mean ± s.e.m. The statistical significance of differences between mean values was assessed by analysis of variance (anova).

Measurement of cytokine induction

Cytokine mRNA was assessed by RT-PCR, as described above. Supernatants from baseline cultures, stimulated cultures (100 ng/ml LPS) and cultures treated with antibodies (MY4 to CD14 and MOPC141, irrelevant) were tested for TNF-α by ELISA (Biotrak, Amersham). Three independent experiments were performed.

Other reagents

LPS (from Escherichia coli, serotype 0111:B4) and PMA (phorbol 12- myristate 13-acetate) were purchased from Sigma-Aldrich (St Louis, MO, USA). Recombinant human interferon (IFN)-γ (specific activity 2 × 107 U/mg) and TNF-α (specific activity 6 × 107 U/mg) were kindly provided by Dr G. R. Adolf (Boehringer Institute, Vienna, Austria); recombinant IFN-α (specific activity 2 × 108 U/mg) was provided by Schering-Plough (Madrid, Spain). Insulin (Actrapid® HM) was purchased from Novo-Nordisk (Bagsvaerd, Denmark).

RESULTS

Human and rat islet cells express CD14 and related molecules TLR4, TLR2 and MD-2

Immunofluorescence

The initial observation made during the characterization of the peri-insular cellular infiltrate in a recent-onset diabetic patient [17] was confirmed in 10 normal pancreases using two MoAbs to CD14 (CRIS6 and MY4), thus ruling out the possibility that CD14 expression in the islets was linked to diabetes. The staining was always diffuse but not bright (Fig. 1a). Double IFL staining with antibodies to GAD that stain all endocrine cell types [28] indicated that CD14 is not expressed selectively. Double staining with antibodies to insulin and glucagon confirmed that both β and α cells express CD14. Scattered CD14 bright cells were present in the exocrine areas of the pancreas, as in other tissues, and both their distribution and morphology corresponded to macrophages. Ductal cells were also slightly positive for CD14, whereas acinar cells were negative. In order to determine whether CD14 was expressed on the cell membrane, sixth-day human islet cell cultures from three different donors were stained for CD14 and insulin. Between 7% and 10% of the cells were β cells (insulin-positive) and most were positive for CD14 (Fig. 1b). The characteristics of the fluorescence and phase contrast images ruled out that these cells were macrophages harbouring phagocyted insulin. In order to confirm these results, freshly dispersed human islet cell preparations from three donors were analysed for cytoplasmic insulin by flow cytometry after staining for surface CD14 and permeabilization. In a representative experiment, a small population (5%) of double-positive cells was detected, thus indicating that approximately half of the insulin-positive (8·9%) cells expressed CD14 (Fig. 2a). Cells from the HP62 epithelial cell line of insular origin and the monocytoid cell line U937 were also stained. CD14 and HLA class I molecules were expressed in both cell lines and although class I staining was stronger than CD14 staining, expression of the latter was clear and reached similar levels in both cell lines (Figs 2d,e). Flow cytometry analysis with calibrated fluorescent beads and islet cells indicated that the average number of CD14 molecules per islet cell was approximately 18 000 (Fig. 2f).

Fig. 1
Human islet cells express CD14. (a) Double IFL staining of pancreatic cryostat section, left to right: CD14 (in green), glutamic acid decarboxylase (GAD) (in red), CD14/GAD overlap, and CD68: macrophages shown in green, insulin shown in red. Original ...
Fig. 2
Flow cytometry analysis of CD14 expression on human islet cells and endocrine cell lines. (a) CD14 expression on freshly isolated and dispersed islet cells: red line CD14, green line normal mouse serum (NMS), grey line isotype-matched control for the ...

RT-PCR confirms the expression of CD14 in the islets and indicates that the other components of the LPS receptor are also expressed

cDNA from purified islets, total pancreatic tissue, HP62 and NES2Y cell lines, THP1 monocytoid cell line and PBLs (positive controls) and Jurkat cells (as negative control) were amplified with CD14 specific primers. The cDNA from islets and pancreatic tissue preparations and from insular and monocytoid cell lines (but not Jurkat cells) gave a positive band that hybridized with the corresponding internal oligoprobe (Figs 3a,,4).4). The detection of CD14 mRNA in islet primary cell cultures and one islet cell line suggested that this mRNA could not belong to passenger leucocytes. CD14 mRNA was also detected by RT-PCR in islets and pancreas from rat (Fig. 4, ‘rat’ panel). CD14 requires other proteins to generate intracellullar signals, and it has been suggested that either TLR4 or TLR2 is the transducing co-receptor while MD-2 enhances the signal. Multiple RT-PCR experiments demonstrated concomitant expression of TLR4, TLR2 and MD-2 molecules in islets, pancreas and insular cell lines as well as in positive controls (PBLs and monocytoid cell line THP1) (Fig. 4). The checkerboard graph in Fig. 4 (bottom) summarizes the results of duplicate experiments that were similar to a Northern analysis (not shown).

Fig. 3
Expression of CD14 and inflammatory cytokines assessed by RT-PCR. (a) Expression of CD14. Top panel: autoradiographs corresponding to the hybridization of the amplified products. Bottom panel: ethidium bromide staining of GAPDH amplification products. ...
Fig. 4
Relative expression of CD14 and associated molecules in islets (human and rat), pancreas and other cell types. Human: top panel, image of the ethidium bromide-stained gel corresponding to GAPDH RT-PCR amplification products used to normalize the results ...

CD14 synthesis and secretion

In order to determine the synthesis and secretion of CD14, culture supernatants from metabolically labelled HP62 and RIN-5F cells were tested for soluble CD14 (sCD14) by immunoprecipitation with the CD14-specific mAb MY4 (Fig. 5a). sCD14 immunoprecipitates were detected in the culture supernatants of both pancreatic cell lines. The sCD14 polypeptides from pancreatic cell lines showed a higher Mr than the sCD14 polypeptides from the monocytic cell line. This different electrophoretic mobility may be caused by a different glycosylation pattern in the sCD14 from insular and monocytic cell lines. Molecular weights higher and lower than that measured for monocyte-derived sCD14 have also been reported for the human B cell- and milk-derived soluble forms of CD14, respectively [10,29]. A time-dependent sCD14 secretory response to LPS has been found in HP62 cells (Fig. 5b, data from two ELISA experiments with similar results, bars representing mean values with a variation lower than 6%).

Fig. 5
Synthesis and release of sCD14 by islet cell lines. (a) SDS-PAGE analysis of sCD14 (arrows) immunoprecipitated with the CD14-specific mAb MY4 using culture supernatants from metabolically labelled cell lines of insular origin (HP62 and RIN-5F) and the ...

β cells express a functional LPS receptor

LPS induces cytokine production in islet cells

Stimulation of HP62 cells with 100 ng/ml LPS for 4 h resulted in de novo transcription of IL-1α and TNF-α mRNA, as detected by RT-PCR (Fig. 3b). The specificity of the band was confirmed by hybridization with the corresponding oligoprobes. IL-1β mRNA was present in baseline conditions but its levels increased after exposure to LPS. These responses are similar to those obtained in the THP-1 monocytoid cell line. As in all other experiments, the amount of cDNA was normalized using the expression of GAPDH, and the reaction was kept within the exponential phase. The measurement of TNF-α levels by ELISA confirmed these results: baseline secretion was 2·63 ± 2·28 pg/ml, and this value rose to 22·78 ± 4·81 4 h after the addition of 100 ng/ml of LPS. This effect was reduced markedly (with final TNF-α levels of 10·50 ± 7·78 pg/ml) by preincubation with the neutralizing MoAb to CD14 (MY4) but not by incubation with an irrelevant MoAb (MOPC-141) (26·34 ± 5·22 pg/ml) (Fig. 3c). Data from three independent experiments.

LPS inhibits glucose-induced insulin release

In a series of previous experiments we had studied insulin secretion at different concentrations of LPS (0·1–1000 ng/ml) in order to ascertain the minimum and maximum dose that could induce an effect on pancreatic islets (data not shown). Because the effect was observed at concentrations of 1–100 ng/ml, we used these concentrations to study the functional action of LPS on islet cells. The effect of LPS on insulin secretion varied markedly depending on the level of glucose but is similar in short-term (90 min) and maintained exposure (36 h) experiments. In short-term cultures, LPS enhanced insulin secretion at a glucose concentration of 5·5 mm glucose while LPS significantly reduced insulin secretion at a glucose concentration of 16·7 mm(Fig. 6a), independently of the dose of LPS. Insulin content was less affected, thus indicating that LPS affects secretion, not synthesis, and that the viability of islets is maintained. Chronic exposure to LPS (36-h cultures) reduced insulin release markedly at a glucose concentration of 16·7 mm but not at 5·5 mm (P < 0·005) in a 90-min assay. This effect was reversible, as islets cultured in LPS for 18 h and then in LPS-free medium for another 18 h recovered the response to 16·7 mm glucose (P < 0·001) at the end of the experiment (Fig. 6c). The insulin content in these islets was not significantly different, thus confirming that LPS influences secretion but not insulin storage (Fig. 6c). The islet perfusion experiments revealed that LPS reduced insulin secretion by about 40% in response to 16·7 mm glucose (data not shown).

Fig. 6
Effect of LPS on insulin production. (a, b) Short-term effects (90 min). Insulin release (a) and content (b): islets cultured at different glucose concentrations (x axis) and LPS: white bars, no LPS; grey bars, 1 ng/ml LPS; black bars, 100 ng/ml LPS. ...

DISCUSSION

The results presented here provide first evidence of the expression of CD14 and other proteins of the LPS-receptor complex in islet cells. The combination of several techniques (immunofluorescence, immunoprecipitation, RT-PCR), substrates (human and rat tissue, fresh and cultured islets and insular cell lines) and specific reagents (antibodies and probes) rule out all common artefacts. Due to limited availability, most functional experiments were carried out using rat islets, which we have shown that also express CD14. In other studies we used islet cell lines which, although being partially dedifferentiated, have the advantage over freshly isolated islets that they had not been exposed previously to endotoxin (as occurs inevitably during islet isolation due to the contamination of collagenase by LPS and are certainly free from tissue macrophages). Endotoxin content clearly decreases after several washes; decreasing from 100 EU/ml after digestion to 2·5 EU/ml after washes and before carrying out the experiments. This concentration did not interfere with the experiments performed[30].

The functionality of the LPS receptor expressed in the islets is supported by three types of evidence:

  1. Modulation of glucose-dependent insulin release, which was enhanced at 5·5 mm glucose and inhibited at 16·7 mm glucose. The almost immediate effect of LPS on insulin release is probably direct because it is too rapid (90 min) to be mediated by cytokines. The effect of maintained (18 h) exposure to LPS may be due to the combined action of LPS and cytokines accumulated in the culture medium. The opposite effects of LPS on insulin secretion at high and low glucose concentrations is interesting but not unique [31].
  2. Transcription of inflammatory cytokine genes. The induction of IL-1α, IL-1β, TNF-α and IL-6 was studied in the HP62 cell line, thus ruling out that the cytokine mRNA could be produced by mononuclear cells remaining in the islets. The predominance of IL-1β over IL-1α is a feature of the human species [32]. The detection of low concentrations of TNF-α in the supernatant of LPS-treated HP62 cells and the threefold decrease of TNF-α levels by the addition of a neutralizing MoAb to CD14 (but not with an irrelevant antibody, MOPC141) confirms that the HP62 cell line produced cytokines in response to LPS via CD14. β cells could contribute to islet inflammation with the synthesis and release of low amounts of cytokines that could act synergically with other cytokines produced by infiltrating cells.
  3. The production of sCD14 in response to LPS is a feature of CD14-positive cells, including some epithelial cells [8,11].

The effects of inflammatory cytokines (i.e. IL-1α, IL-β and IFN-γ) on islet β cells has been investigated extensively, thus leading to a widely accepted model according to which the destruction of these islet cells in type I diabetes would result from local release of IL-1 by infiltrating macrophages [33]. It has been suggested that the induction of iNOS expression by β cells in response to IL-1 is a key factor for cytokine-dependent islet toxicity [34,35]. We have not found any islet cell damage, and the reduction in insulin release was not associated to a concomitant decrease in insulin content, thus indicating that insulin synthesis, and most probably cell viability, was maintained despite exposure to LPS. The LPS concentrations used in our experiments were very moderate (up to 100 ng/ml), similar to those used to stimulate macrophages [36] and well below those used by authors who have shown the cytotoxic effect of the combination of TNF-α plus LPS (5 µg/ml) on the islets [37]. Our data indicate that LPS may act directly on the β cells and this may help to interpret in vitro and in vivo experiments that lead to the suggestion that LPS acts through cytokine production.

The finding that islet β cells express endotoxin receptor raises the question of its physiological role. This question applies to a growing number of cell types that have been shown to express CD14 [16]. Other endocrine cell lineages also respond to LPS. For instance, the ATt-20 pituitary cell line produces macrophage migration inhibitory factor (MIF) when stimulated with LPS [38] in conditions (serum dependency, LPS levels of 1–100 ng/ml) that suggest that they express the LPS receptor. There is also growing evidence that the endocrine and the immune system interact at several levels [39]: a regulatory circuit that involves the cytokines that trigger the acute phase reaction i.e. IL-1, IL-6 and TNF-α and the activation of the pituitary–adrenal axis has been described [40].

We have found that relatively low endotoxin concentrations enhance insulin secretion. A possible physiological justification would be that the adjustment to increased metabolic demands typical of acute infections requires an increase in glucose uptake by most cells. Later, after the development of septic shock, a reduction in insulin production may be needed to redirect glucose to the most essential organs (brain and heart) which do not need insulin to uptake glucose. Recent evidence suggests that this effect is probably regulated by MIF [41]. It is also possible that secretion of CD14 by many cell types helps to provide enough sCD14 to bind and remove LPS from the bloodstream and also to opsonize bacteria. It has been suggested that the LPS/LBP/sCD14 complexes can activate cells that express little or no CD14, such as the endothelial cells [42]. We have found that islet cells respond to LPS not only by reducing insulin secretion but also by initiating the transcription of cytokine genes. CD14 in β cells may have the same function as in other non-myeloid cells: a PRR that triggers the activation of a cell adaptation program to stress or imminent cell injury, and this includes the production of heat shock proteins and inflammatory cytokines [43].

Islet transplantation is a procedure that has a low success rate in humans: about 10% of insulin independence after 1 year [44]; however, a recent report using non-steroid immunosuppression is promising [45]. Most failures are due to primary non-function. The expression of CD14 and related molecules by islet cells may help to explain the failure of grafted islets to regain function after transplantation. Contrary to other tissue preparations used for transplantation, islets undergo a complex and long isolation procedure during which they are exposed to collagenase, an enzyme that contains endotoxin [30]. Thus, transplanted islets probably undergo a transient inhibition of insulin release in response to glucose and, more importantly, produce cytokines that could contribute to inhibit insulin secretion further and enhance an inflammatory process that leads easily to early rejection.

Other recent observations add significance to the expression of CD14 by islet cells: respiratory syncytial virus [46] and shock protein 70 and 60 [47,48] stimulate cells through CD14 and TLR4. Although it may be premature to suggest that CD14 and TLRs contribute to make the islets susceptible to viral infection, the expression of the LPS receptor complex places the β cells at the junction of the innate and adaptive immune response [49], a postulated checkpoint in the development of autoimmune diseases [50]. Finally, some experiments have shown that LPS confers diabetogenic potential to the T cell repertoire of a TCR transgenic NOD mice [51].

In summary, the demonstration that islet cells express endotoxin receptor and that endotoxin regulates the production of insulin and inflammatory cytokines opens new possibilities for the interpretation of the initial process that leads to diabetes and may help to understand the causes of islet cell transplantation failure.

Acknowledgments

This work was supported by grants from the ‘Fondo de Investigaciones Sanitarias, FIS’ (Projects 96/0639 and 99/1066 to M.V.P. and 99/0837 to R.G.). N.S. and J.F.-A. were supported by the Comissió Interdepartamental de Recerca i Tecnologia (CIRIT), Generalitat de Catalunya (action SGR 00105 and a direct contract, respectively), F.V. was supported by a personal fellowship of the Fundació Catalana de Transplantament and M.V. was supported by postdoctoral fellowship no. 396161 of Juvenile Diabetes Foundation International. We thank the colleagues cited in the text for their donations of monoclonal antibodies and other reagents. We also thank Dr R. Casamitjana (HCP, Barcelona) for RIA determinations. We acknowledge the help of M. Martí, L. Alcalde and P. Armengol in collecting materials, Dr M. Juan in molecular biology techniques and M.A. Fernandez in flowcytometric techniques.

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