Can CD44 Be a Mediator of Cell Destruction? The Challenge of Type 1 Diabetes.

CD44 is a multi-functional receptor with multiple of isoforms engaged in modulation of cell trafficking and transmission of apoptotic signals. We have previously shown that injection of anti-CD44 antibody into NOD mice induced resistance to type 1 diabetes (T1D). In this communication we describe our efforts to understand the mechanism underlying this effect. We found that CD44-deficient NOD mice develop stronger resistance to T1D than wild-type littermates. This effect is not explained by the involvement of CD44 in cell migration, because CD44-deficient inflammatory cells surprisingly had greater invasive potential than the corresponding wild type cells, probably owing to molecular redundancy. We have previously reported and we show here again that CD44 expression and hyaluronic acid (HA, the principal ligand for CD44) accumulation are detected in pancreatic islets of diabetic NOD mice, but not of non-diabetic DBA/1 mice. Expression of CD44 on insulin-secreting β cells renders them susceptible to the autoimmune attack, and is associated with a diminution in β-cells function (e.g., less insulin production and/or insulin secretion) and possibly also with an enhanced apoptosis rate. The diabetes-supportive effect of CD44 expression on β cells was assessed by the TUNEL assay and further strengthened by functional assays exhibiting increased nitric oxide release, reduced insulin secretion after glucose stimulation and decreased insulin content in β cells. All these parameters could not be detected in CD44-deficient islets. We further suggest that HA-binding to CD44-expressing β cells is implicated in β-cell demise. Altogether, these data agree with the concept that CD44 is a receptor capable of modulating cell fate. This finding is important for other pathologies (e.g., cancer, neurodegenerative diseases) in which CD44 and HA appear to be implicated

Insulin-producing β cells from WT NOD females display enhanced susceptibility to apoptosis.

<p>A) Enhanced caspase-3 activity is detected in cell extracts of WT than in those of CD44-deficient pancreatic islet cells. Pancreatic islet cells were freshly removed from WT and CD44-deficient pre-diabetic NOD females. Cell extracts from islets were subjected to Western blot analysis, using anti-cleaved caspase-3 antibody. A representative Fig of three experiments. (B and C) Pancreata were harvested from 10-weeks-old normoglycemic WT and CD44-deficient NOD females (n = 5 in each group). β-cell apoptosis was assessed using TUNEL assay (green) and co-staining for insulin (red) and DNA (blue). B) Apoptosis was calculated as percent of TUNEL positive nuclei (+/- SEM) in insulin-positive cells out of the total number of insulin-positive cells per mouse. WT (white bar, ~17,560 total β-cells); CD44-deficient (black bar, ~14,800 total β-cells). C) Pictures are representative images of islets from WT (left panel) and CD44-deficient NOD females (right panel). Original images were taken at a magnification of x40. Inset images were digitally increased x5. White arrowheads point to apoptotic β-cells.</p

CD44-null NOD females display relative resistance to T1D: role of inflammatory cells.

<p>A) and B) Development of T1D in the spontaneous (A) and cell transfer (B) models of WT and CD44-deficient mice was monitored by measuring blood glucose. In the transfer model, irradiated CD44-deficient and WT young (6–8 weeks-old) male recipients were respectively transplanted with splenocytes from CD44-deficient and WT diabetic females. Percentage of diabetes-free mice (showing <250 mg/dL blood glucose) was recorded versus time. Statistical analysis by Breslow; <i>A</i>, <i>P</i> < 0.05; <i>B</i>, <i>P</i> < 0.005. C) The invasion capacity of infiltrating cells derived from WT (n = 6) and CD44-deficient (n = 6) NOD females (spontaneous model) was measured as indicated in <i>Materials and Methods</i>. A total of 754 pancreatic islets in each mouse group were scored by an uninformed observer. The percentage of islets showing each one of the infiltrating scores was calculated for each mouse, and the average values are presented. Islets from CD44-deficient mice display higher percentage of infiltrating scores (2, 3 and 4) than islets from wild type mice. <i>P</i> < 0.0001 by Pearson's χ2 test comparing the distribution of scores in the two mouse groups. D) Wild type or CD44-deficient pre-diabetic spleen cells were added to the top compartment of transwell migration chamber, separated by HA-coated filter (10 μg/filter) from the bottom compartment, and the % of cells migrating through the filter toward the chemoatractant in the bottom compartment was calculated by flow cytometry. Statistical analysis by Student’s-<i>t-</i> test. Accumulated data of 4 experiments (n = 8; error bars, SEM).</p

CD44 involvement in T1D.

<p>Three CD44-dependent pathways affect the fate of T1D in NOD mice. I. CD44 involvement in inflammatory cell migration (blue). Inflammation upregulates CD44 on the islet-infiltrating cells. The firm adhesion of the CD44 receptor to the HA substrate slows the motility of the inflammatory cells on the endothelium. II. CD44 involvement in peripheral insulin sensitivity and glucose uptake (green). CD44 expression on peripheral tissue, increases insulin sensitivity and glucose uptake by, for example, muscle and liver cells, resulting in reduced hyperglycemia. Compensation for insulin-deficiency (see pathway III) may explain this event. III. CD44 involvement in β cell apoptotic death (red). Inflammation-induced cytokines up-regulate CD44 expression on β cells. CD44 expression on β cells is associated with increased β cell dysfunction and susceptibility to apoptosis, which could be triggered by the binding of HA fragments to CD44 receptor. However, large HA fragments could interfere with the detrimental effect of LMW-HA on β cells. The β cell dysfunction and susceptibility to apoptosis is indicated by increased iNOS induction and subsequent NO production, increased caspase-3 activity (Western blot, not shown), reduced glucose-stimulated insulin secretion, and reduced insulin content. As an outcome of β cell demise, hyperglycemia is detected, implying the development of T1D. Broken arrows and question marks represent pathways and factors that are yet to be established.</p

CD44-deficient pancreatic islet cells retain their functionality under pro-inflammatory conditions.

<p>(A and B) <i>In vitro</i> pro-inflammatory conditions. A) Griess assay. Wild type and CD44-deficient pancreatic islet cells were incubated with medium (-) or Cytomix for 24 and 48 h. Nitrite release was assessed by Griess assay (n = 3). Inset: Cell extracts from pancreatic islets described in (<i>A</i>) were subjected to Western blot analysis, using anti-iNOS antibody. A representative Fig of three experiments. B) Glucose-stimulated insulin secretion (GSIS). Pancreatic islet cells from WT and CD44-deficient NOD mice were incubated with medium (-) or Cytomix for 48 h and then stimulated with 3.3 or 16.7 mM glucose for additional one hour and their ability to secrete insulin was measured by ELISA. Inset: Insulin content in each one of the samples (n = 4–5) (error bars, SEM). Statistical analysis for A and B by standard two-tailed Student’s <i>t</i>-test. <i>A</i>, one representative experiment of five. <i>B</i>, one representative experiment of two. (C and D) <i>Ex vivo</i> pro-inflammatory conditions. C) Freshly isolated pancreatic islet cells from WT and CD44-deficient pre-diabetic NOD females were processed as described in (<i>B</i>) (n = 6–8) (error bars, SEM). D) Content of insulin in pancreatic islets of WT and CD44-deficient pre-diabetic NOD females was analyzed by ELISA. Statistical analysis in <i>C</i> and <i>D</i> by standard two-tailed Student’s <i>t</i>-test. The statistical analysis in <i>C</i> shows that the <i>ex vivo</i> insulin release of CD44-deficient pancreatic islets at 12 and 14 weeks is significantly higher (p<0.05) than the insulin release of the corresponding WT cells.The statistical analysis in <i>D</i> shows that the insulin content in pancreatic islets of 14 wks-old pre-diabetic WT mice is significantly different (<i>P</i> < 0.05) from all other samples. (n = 6–8). In <i>C—D</i>, at least one representative experiment of two.</p

CD44 expressed in the recipient’s tissue rather than on the infiltrating-cells enhances the development of T1D in the cell transfer model.

<p>A) CD44-deficient non-diabetic NOD male recipients were reconstituted with inflammatory spleen cells derived from WT (CD44^+/+→CD44^-/-; marked #1) or CD44-deficient (CD44^-/-→CD44^-/-; marked #2) diabetic NOD female donors. In addition, WT non-diabetic NOD male recipients were reconstituted with inflammatory spleen cells derived from WT (CD44^+/+→CD44^+/+; marked #3) or CD44-deficient (CD44^-/-→CD44^+/+; marked #4) diabetic NOD female donors. Inflammatory cells are marked by arrows and local islet cells by arrowhead. The pancreases of all 4 combinations were removed after detection of diabetes or 200 days after cell transfer. Doing so, pancreases that were harvested approximately at the same time, were stained with anti-CD44 mAb. B) The % of diabetes-free mice (exhibiting less than 250 mg/dL urine glucose) was determined in corresponding four mouse groups, described in <i>A</i>. Statistical analysis by Breslow: #2 versus #3, <i>P</i> < 0.01; #1 versus #2 (follow up 75 to 200 days), <i>P</i> < 0.03; #3 versus #4 (follow up 20 to 75 days), <i>P</i> < 0.03.</p