The role of chemokines in type 1 diabetes: as assessed by RT-PCR

Type 1 diabetes is an autoimmune disease, characterised by immune infiltration into the islets of Langerhans, resulting in the destruction of insulin producing b-cells. Over recent years, evidence has been collected on the important role of chemokines in the recruitment of immune cells leading to the pathology of this disease. In this review we discuss the findings on the role of chemokines, as obtained from animal studies. We will focus on the quantification of chemokines and chemokine receptors, making use of the innovative real-time quantitative PCR technique.

Abbreviations

FW – Forward primer
RV – Reverse primer
TP – TaqMan® probe
SYBR, SybrGreen™ assay.
a – Amplicon length in base pairs
b – Genbank accession number of cDNA and corresponding gene, more information is available at www.ncbi.nlm.nih.gov

Real-time PCR: an introduction

The real-time PCR (RT-PCR) technique is an especially sensitive and accurate technique for the quantification of mRNA levels in cells, tissues and tissue biopsies. Since its invention in the early nineties,1,2 its use has increased exponentially and it is now the method of choice for quantification of mRNA levels. For the quantification of chemokine and chemokine receptor levels specifically, it is ‘the golden standard’, as these factors are often expressed at very low levels in cells or tissues of interest. Currently different DNA polymerases and a wide range of different detection chemistries are available, the original method developed for the quantification of mRNA made use of the intrinsic capacity of the Thermus Aquaticus (Taq) polymerase to amplify DNA and simultaneously, the 5’ nuclease activity of this enzyme to cut an internally annealing dual-labelled fluorescent probe. This degradation of the dual-labelled probe gave rise to a fluorescent emission, which could be measured during the course of the PCR reaction1,2,3 this is the ‘so-called’ specific detection method. A second widely used method makes use of non-specific detection chemistry, such as SYBR Green. In this case, all dsDNA amplified during the reaction is detected.

Therefore, it is very important to include additional controls to distinguish between the target sequence of interest on one hand, a-specific products or primer dimers on the other4.

In both applications, PCR amplification and data acquisition (by measuring the increase in fluorescent emission as the PCR reaction proceeds) is performed in one single step, since fluorescent emission is measured continuously during the PCR reaction, in real-time. In case of non-specific chemistries, a post-PCR melting curve analysis needs to be performed. Quantification of the target of interest is based the increase in fluorescent emission as a direct measure for the amount of amplified PCR product5.

Different factors have lead to the widespread use of real-time PCR assays, being present as a routine technique on almost every laboratory bench. First of all, as opposed to the classical PCR which only resulted in ‘qualitative’ data, it is a ‘quantitative’ method, giving a very good estimation of initial mRNA copy numbers. Secondly, PCR amplification and detection are performed in a single step, resulting in short analysis time and high throughput. Thirdly, quantification is possible over a wide dynamic range. Fourthly, reproducibility and sensitivity of the method are very high, making the method suitable for analysis on very small amounts of cells or tissue samples. This enhanced sensitivity is of particular interest for research and diagnosis in the field of immunology, where cytokines and chemokines are often expressed at very low levels and only small amounts of sample material can be used6,7. Finally, its widespread use has been facilitated by the introduction of second generation instrumentation as well as the design of alternative chemistries; with the natural competition between different commercial suppliers making the technique affordable for almost every scientific and clinical laboratory.

Studies in animal models

Many studies in animal models, most of them performed in the non-obese diabetic (NOD) mouse, have aimed at gaining a better insight into the role of chemokines in the initiation or development of type 1 diabetes8. Early studies in mice have already shown an important role for chemokines in the recruitment of effector T-cells9,10,11 and regulatory T-cells12 to the islets of Langerhans. Furthermore, the CC chemokine gene family is located in the Idd4 diabetes susceptibility locus13, providing genetic evidence for the involvement of chemokines in the pathology of the disease. Based on these findings it is very likely that different chemokines are associated with T-cell differentiation and recruitment as well as with diabetes susceptibility in NOD mice.

The site of immune attack, namely the islets of Langerhans, has been the subject of many studies investigating the expression profile of chemokines, performed in different mouse models.

A first study identified the expression of a whole range of chemokines in islets from NOD-SCID mice, including CXCL10 (IP10), CCL22 (MDC; macrophage-derived chemoattractant), CCL21 (SLC; secondary lymphoid tissue chemokine), CCL3 (MIP1a), CCL17 (TARC; thymus and activation regulated chemokine) and CCL2 (MCP1)14. In addition, high levels of CCL2 (MCP1), CXCL10 (IP10), CCL20 (MIP3a) and the cytokine IL15 were measured in pancreatic islets from pre-diabetic NOD mice, as well as in islet isografts after transplantation into diabetic NOD mice15,16. Another interesting model is the RIP-GP mouse model, a transgenic mouse expressing the glycoprotein of the lymphocytic choriomeningitis virus (LCMV) under the control of the rat insulin promoter17. These mice develop severe insulitis upon infection with LCMV. Different chemokines, namely CXCL10 (IP10), CXCL9 (MIG), CCL2 (MCP1) and CCL5 (RANTES), were highly induced in the pancreatic islets of these mice, concurrent with insulitis development. Also, the chemokine receptors CCR5, CCR2 and CXCR3 were detected specifically in the islets of these transgenic mice, but not in control islets13,18. Interestingly, most of these studies again made use of the real-time PCR technique to measure chemokine transcript levels.

Neutralisation of CXCL10 (IP10) suppresses the development of diabetes in NOD mice, at least after cyclophosphamide induced disease acceleration in NOD mice, through enhanced b-cell proliferation in insulitis19, indicating an important role for this chemokine in the development of diabetes. In contrast, when expressed outside of the pancreas, IP10 can suppress diabetes. This was shown by Christen et al., both in NOD and in a transgenic RIP-LCMV model of autoimmune diabetes. A selective expression of IP-10 was seen in the pancreatic draining lymph node20. In both cases, infection with the LCMV virus whilst the auto aggressive process is already in progress was able to arrest type 1 diabetes progression. This study highlights the importance of correct location, time and expression levels20. Transgenic mice with b-cell specific expression of IP10 did not exhibit spontaneous diabetes when crossed with the RIP-LCMV mice. However, the onset of virus induced diabetes was much earlier in these mice, providing evidence for the involvement of chemokines in the attraction of lymphocytes to the inflamed target site21. Moreover, a direct role for IP10 was convincingly shown in CXCR3 knockout mice, which showed a delayed diabetes onset and prolonged islet graft survival. A similar finding was observed making use of alpha-rIPa (anti-IP10) mAb therapy18,22. In addition, an association between increased IP10 and MCP1 levels was consistent, with a decrease in apoptosis and preserved glucose stimulated insulin release in IRF1 deficient mice23.

An important role in islet infiltration is also evident for CCL2 (MCP1), as shown by different studies24. It has been suggested that MCP1 plays a role in the initiation of type 1 diabetes, contributing to early islet infiltration by attracting lymphocytes. This was shown in the NOD mouse model25. In transgenic mice over-expressing MCP1 and under the control of a rat insulin promoter, a direct role for MCP1 in pancreatic monocytic infiltration was shown26.

CCL3 (MIP1a) is another chemokine shown to play a role in type 1 diabetes development. In NOD mice deficient in MIP1a (NOD.MIP1a knockout), diabetes incidence was significantly reduced and delayed. In addition, the ratio of MIP1a /MIP1‚ in the pancreas appeared to be important during the initial stages of islet mononuclear cell infiltration in determining the nature of insulitis progression in NOD mice25.

Concluding from these studies, chemokines clearly plays a major role in b-cell destruction. The advances recently obtained in this field have been greatly aided by the development of real-time RT-PCR assays for the detection of chemokines in the islets of Langerhans. Indeed, this technique made it possible to investigate and quantify a multitude of chemokines expressed in the islets. Consequently, this has aided to our understanding of the pathology of type 1 diabetes. Combined with numerous studies, deleting or over expressing one chemokine by use of knockout and transgenic mice, or by use of systemic administration of chemokines, or their specific antibodies, it is clear that the expressed chemokines, indeed play an important role in the development of type 1 diabetes. Most probably, however, it will be the cooperation of many cytokines and chemokines which is crucial in mediating the initiation and development of insulitis and diabetes outcome – in mice as well as in humans.

Conclusions

The invention of real-time PCR has not only enormously simplified the quantification of DNA and RNA, it has also resulted in much more accurate and reliable data. This had a great impact in the field of molecular research and diagnostics, since enormous amounts of data could be obtained within a very short research time. In fundamental research on type 1 diabetes, the technique has significantly improved our understanding of b-cell destruction, for instance by the high throughput analysis of chemokine expression levels at the site of immune attack, ie. the islets of Langerhans. The decreased costs for thermal cycling instruments, as well as for the necessary reagents to perform the reactions, have obviously assisted in its rapid, not to say exponential, increase in use. It is beyond any doubt that this technique has become the ‘golden standard’ for the detection and quantification of DNA and RNA in fundamental research, pre-clinical and clinical settings; including the quantification of chemokines.

Although real-time PCR assays by themselves are characterised by high precision and reproducibility, the accuracy of the data obtained relies largely on several other factors. Indeed, it is not sufficient to simply extend one’s knowledge of classic endpoint PCR. Many other controls have to be included to obtain accurate results. Issues to be taken into account for instance are sample preparation, quality of the RNA, choice of a relevant housekeeping gene, correct normalisation of the samples, experimental setup and accurate data analysis5,27.

Most probably, since RT-PCR has become a routine technique present in almost every laboratory, its use is expected to increase even more during the coming decade. However, it remains crucial that specific controls as to the validity of each specific assay are taken into account. Apart from this, as we have reached the post-genomic area, one has to realise that when making use of this technique, no conclusions can be made to the functional implications at the protein level. Combining RT-PCR results with proteomic results, will certainly unearth a more complete view of the dynamic alterations taking place in a cell or tissue of interest under certain physiological or pathological conditions.

It is to be expected that in the field of proteomics analysis, with the major advances being made during the last decade on highly sophisticated mass spectrometry instrumentation, the analysis of translational and post-translational control (eg. post-translational modifications, proteolytic cleavage) will evolve rapidly and enlarge our view on the function and importance of chemokines in type 1 diabetes. These, in combination with RNA based techniques as discussed herein, will greatly aid in filling the gaps that still remain in our understanding of this complex disease.

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