Immune-mediated processes have been implicated in the pathogenesis of diabetic polyneuropathy. Langerhans cells (LCs) are the sole dendritic cell type located in the healthy epidermis and exert tolerogenic immune functions. We aimed to determine whether alterations in cutaneous LC density and intraepidermal nerve fiber density (IENFD) are present in patients with recently diagnosed type 2 diabetes. Skin biopsy specimens from the distal leg from 96 type 2 diabetic patients and 75 healthy control subjects were used for quantification of LC density and IENFD. LCs and IENFs were labeled using immunohistochemistry. Nerve conduction studies, quantitative sensory testing, and neurological examination were used to assess peripheral nerve function. LC density was markedly reduced in the diabetic group compared with the control group, but did not correlate with reduced IENFD or peripheral nerve function. Multivariate linear regression analysis revealed a strong association between LC density and whole-body insulin sensitivity in women but not in men with diabetes. Prospective studies should establish whether the pronounced reduction of cutaneous LCs detected in recently diagnosed type 2 diabetes could promote a cutaneous immunogenic imbalance toward inflammation predisposing to polyneuropathy and foot ulcers.
Recent evidence suggests the involvement of inflammatory processes in the pathogenesis of type 2 diabetes (T2D) (1) and its complications such as polyneuropathy (2). Diabetic neuropathy is associated with extensive morbidity, increased mortality, and reduced quality of life (3). A distal leg skin biopsy specimen with quantification of intraepidermal nerve fiber density (IENFD) is a reliable and efficient technique to assess the diagnosis of small fiber neuropathy (SFN) (4). However, the pathophysiological processes triggering the loss of IENFs in diabetic neuropathy are unknown. Langerhans cells (LCs) are the sole dendritic cells located in the healthy epidermis. IENFs have been shown to have an immune-modulatory effect on LCs (5). This finding indicates a close relationship between the nervous system and the function of distinct immune system components. In patients with type 1 diabetes, cutaneous LC density was markedly reduced immediately at diabetes onset, but not in those with a known diabetes duration of 6 months (6). An increase in LC density has been reported in diabetic patients with painful SFN, but not in glucose-tolerant individuals with painful or painless SFN (7) or those with neuropathic pain due to postherpetic neuralgia (8). However, these studies evaluated rather small samples of subjects. Whether LC density is altered in relation to IENFD early during the course of T2D is unknown. Early detection of putative neuroimmune alterations could be useful in developing strategies to prevent clinically advanced neuropathy and foot ulcers. We therefore aimed to assess LC density and IENFD and their relationship to metabolic factors in patients with recently diagnosed T2D.
Research Design and Methods
The study was conducted in accordance with the Declaration of Helsinki and was approved by the ethics committee of the Heinrich Heine University, Düsseldorf. All participants provided a written informed consent. Healthy control subjects (n = 75) and patients with recently diagnosed T2D (n = 96) were studied. Subjects with T2D were participants of the prospective German Diabetes Study (GDS), which evaluates the long-term course of diabetes and its sequelae (9). Inclusion criteria for entry into the GDS were type 1 or type 2 diabetes, known diabetes duration ≤1 year, and 18–69 years of age. Exclusion criteria were secondary diabetes, pregnancy, severe diseases (cancer), psychiatric disorders, immunosuppressive therapy, limited cooperation ability, and neuropathy from causes other than diabetes. Inclusion criteria for the control group were age ≥18 years, and exclusion criteria corresponded to those applied to the diabetic group, except for an abnormal oral glucose tolerance test result (10) and neuropathy from any cause.
Hyperinsulinemic Euglycemic Clamp
The hyperinsulinemic euglycemic clamp was performed in diabetic patients according to the Botnia protocol, as previously described (11). In brief, after a 60-min intravenous glucose tolerance test, study participants received a 10-min insulin bolus (40 U/h) and continuous insulin infusion (40 mU × m2 × min−1). Plasma glucose was adjusted with 20% glucose enriched with 2% d-[6,6-2H2]glucose to 5.0 mmol/L. Whole-body glucose disposal (insulin sensitivity) was calculated as the M value, as previously reported (12).
Peripheral Nerve Function
Peripheral nerve function tests were performed as previously described (13). Motor nerve conduction velocity was measured in the median and peroneal nerves, and sensory nerve conduction velocity was determined in the median and sural nerves at a skin temperature of 33–34°C using Nicolet VikingQuest surface electrodes (Natus Medical, San Carlos, CA). Quantitative sensory testing was evaluated by vibration perception threshold at the second metacarpal bone and medial malleolus using the method of limits (Vibrameter; Somedic, Stockholm, Sweden) and by thermal detection thresholds including warm and cold thresholds at the thenar eminence and dorsum of the foot using the method of limits (TSA-II NeuroSensory Analyzer; Medoc Advanced Medical Systems, Ramat Yishai, Israel). Neurological examination was performed using the Neuropathy Disability Score and the Neuropathy Symptom Score (14).
Skin Biopsy and Tissue Fixation
Three-millimeter skin punch biopsy specimens were taken under local anesthesia from the left lateral calf, ∼10 cm proximal to the lateral malleolus. The tissue was fixed with 2% periodate-lysine-paraformaldehyde at 4°C for 24 h, rinsed twice for 10 min with 0.1 mol/L Sorensen buffer, and incubated in 33% sucrose for 3 h. After cryoprotection with 0.02 mol/L Sorensen buffer containing 20% glycerol at 4°C overnight, tissue was stored at −80°C (4,15).
Serial sections of skin biopsy specimens at a thickness of 50 µm for IENF detection and at 10 µm for LC detection were cut perpendicular to the skin.
Staining of IENF was performed following the free-floating method, as described before (15) with some modifications. In brief, after blocking, sections were incubated with a rabbit anti-PGP9.5 antibody (Millipore, Temecula, CA) and a biotinylated anti-rabbit IgG antibody (Vector Laboratories, Burlingame, CA) for 1 h, followed by incubation for 1 h with the Vector ABC kit and for 3 min with the Vector SG substrate kit. All steps were performed at room temperature.
For the detection of LCs, sections were blocked, incubated with the mouse anti-Langerin (12D6) antibody (Abcam, Cambridge, U.K.) overnight and a biotinylated anti-mouse IgG antibody (Vector Laboratories) for 45 min, followed by incubation with Vector ABC and Vector SG substrate. All steps were performed at room temperature.
Quantification of IENFD and LC Density
A method adopted by the European Federation of Neurological Sciences was used (16) for the quantification of IENFD. Individual IENFs from four cross-sections per subject were visually counted along the length of the epidermis using a Leica DMRBE inverted microscope (Leica, Wetzlar, Germany) equipped with an Olympus DP73 digital color camera (Olympus, Hamburg, Germany), and cellSens v1.7 imaging software (Olympus Europa, Hamburg, Germany). Only the IENFs crossing the dermal-epidermal border were counted.
Two sections per subject were used for the quantification of LC density, and the mean was calculated. LCs in the epidermis were visually counted using the ×40 objective and cellSens software.
For normally distributed data, parametric tests (t test or Pearson product-moment correlation), otherwise nonparametric tests (Mann-Whitney U test or Spearman rank correlation) were applied. To determine possible correlations between two variables, multiple linear regression analyses were performed. Continuous data are expressed as median and interquartile range or mean ± SD. Categorical data are given as percentages of subjects. The level of significance was set at α = 0.05.
Anthropometric, demographic, and neurological measures in the groups studied are reported in Table 1. Diabetic patients had a higher BMI than control subjects: 28 (29.2%) of 96 patients were overweight and 60 (62.5%) were obese. Peroneal motor nerve conduction velocity, sural sensory nerve conduction velocity, and cold thermal detection thresholds were lower, whereas malleolar vibration perception threshold and Neuropathy Symptom Score were higher in the diabetic patients than in the control group. In the diabetic patients, HbA1c was 6.3 (1.1)% or 45.4 (7.8) mmol/mol, M value was 5.04 (3.05), and known duration of diabetes until skin biopsy was 12.0 (12.0) months. The percentages of patients receiving diet only, oral glucose-lowering drugs, and insulin were 44.8, 51.0, and 4.2%, respectively.
IENFD was lower in the diabetic group than in the control group (7.66 [4.06] vs. 9.22 [4.32] fibers/mm; P = 0.002) (Fig. 1A). The analyses of the LC density (Fig. 1B) revealed a pronounced LCs reduction in diabetic patients compared with control subjects (387  vs. 563  cells/mm2; P < 0.0001). The difference in LC density between the groups remained significant after adjustment for age, sex, BMI, and smoking (β = −0.313; P < 0.0001).
Correlation analyses did not reveal a group- or sex-specific relationship between IENFD and LC density. However, a sex-specific correlation of LC density with the M value as a measure of insulin sensitivity was observed in diabetic women (R2 = 0.435; P < 0.0001) (Fig. 2A). The correlation remained strongly positive after adjustment for age, BMI, smoking, known diabetes duration, and HbA1c (β = 0.749; P = 0.001) (Table 2). In contrast, LC density was not correlated with M value in diabetic men (Fig. 2B). There was no difference between diabetic men and women in M value (5.09 [2.95] vs. 5.04 [4.07] mg/kg × min) and HbA1c (6.3 [1.1] vs. 6.3 [1.2]% or 45.4 [7.6] vs. 45.4 [8.6] mmol/mol).
There is evidence to suggest that chronic subclinical inflammation is implicated in the pathogenesis of T2D (1,17,18). We previously demonstrated in a population-based study that subclinical inflammation is also associated with diabetic polyneuropathy (2). However, whether components of the immune system are related to IENF loss in diabetic SFN is unknown. Epidermal LCs are the sole dendritic cells of a healthy epidermis, and their function is in close relationship with IENFs (5). Recently, it became evident that LCs, which essentially represent a subset of dendritic cells, have a unique feature of maintaining immune tolerance (19,20). We describe here a striking reduction of LC density in patients with recently diagnosed T2D. Because LCs exhibit anti-inflammatory properties, the reduction of epidermal LC density should result in a shift to a proinflammatory cutaneous environment. If inflammation is involved in diabetic SFN, one would expect a direct relationship between LC density and IENFD. However, we did not find such an association in this population. This suggests that, at least in recently diagnosed T2D, reduced LC density may not affect IENFD. This notion is in accordance with a recent experimental study reporting that the effect of diabetes on LC proliferation and maturation in rats was independent of effects on cutaneous innervation (21). However, it is possible that there is a time-dependent impact of reduced LC density on small nerve fibers that can only be detected in a long-term prospective study. On another note, diabetes and diabetic polyneuropathy predispose to foot infections and ulcers. It is tempting to speculate that foot infections and ulcers could be predicted by the distinct loss of LCs observed herein.
The results of the current study differ from those of a recent study reporting an increase in LC density in a small group of diabetic patients with painful SFN (7). Moreover, the presence of diabetes in that study was associated with increased LC density, and IENFD was inversely related to LC density. In contrast, we demonstrate that recently diagnosed T2D is characterized by a marked reduction of LCs in the lower limbs that is not related to IENFD. Interestingly, the mean LC number in the subjects with diabetes studied by Casanova-Molla et al. (7) was similar to the LC count in the current study. However, LC density was considerably lower in their control subjects than in ours. We have no explanation for this discrepancy, but the LC number established in our control subjects corresponds to the findings of several previous studies (6,22).
An unexpected finding was the strong positive correlation between the M value as a measure of insulin sensitivity and LC density in diabetic women. Previous work has shown that inflammatory markers were stronger predictors of incident T2D in women than in men (23). One reasons for this finding could be the sexual dimorphism in the immune response in humans; for example, females produce more vigorous cellular and humoral immune reactions and are more resistant to certain infections (24). LCs maintain immune tolerance in normal skin by selectively and specifically inducing activation and proliferation of skin resident regulatory and memory T cells (19,20). Inflammation is known to associate with insulin resistance (25). The reduction of LC density could result in an impairment of skin tolerance maintenance and, therefore, to a cutaneous proinflammatory environment shift. This could be one possible explanation for the correlation of LC density with insulin sensitivity in diabetic women.
The strength of the current study is the large number of subjects available for morphometric analyses. However, the study has also two limitations. First, because the M value was not available in the control group, we could not determine whether insulin sensitivity associates with LC density also in glucose-tolerant women. Second, the cross-sectional nature of the present analysis did not allow us to assess the predictive value of the described reduced LC density and IENFD. However, prospective analyses in the present diabetic and control groups will be performed at the 5-year and 10-year follow-up of the GDS.
In conclusion, we demonstrate that patients with recently diagnosed T2D show a striking reduction of LC density. In women, LC density is associated with insulin sensitivity. Because LCs promote cutaneous immune tolerance, long-term prospective studies will determine whether their reduction could be a predisposing factor for cutaneous infection and ulceration in diabetic patients.
M.R. and D.Z. share senior authorship.
Acknowledgments. The authors thank the staff of the GDS, especially M. Behler, B. Ringel, and M. Schroers-Teuber, for their excellent technical support.
Funding. The GDS was initiated and financed by the German Diabetes Center, which is funded by the German Federal Ministry of Health (Berlin, Germany); the Ministry of Innovation, Science, Research and Technology of the state North Rhine-Westphalia (Düsseldorf, Germany); and grants from the German Federal Ministry of Education and Research to the German Center for Diabetes Research.
Duality of Interest. No potential conflicts of interest relevant to this article were reported.
Author Contributions. A.S. and D.Z. designed the experiments, analyzed data, and wrote the manuscript. J.B., I.Z., and K.J. conceived the experiments. J.W., H.A.-H., and M.R. contributed to discussion and reviewed and edited the manuscript. D.Z. is the guarantor of this work and, as such, had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.