Natural killer cell and islet killer cell activities in Type 1 (insulin-dependent) diabetes

Diabetologia, Jun 1986

Summary Peripheral blood mononuclear cells from 20 Type 1 (insulin-dependent) diabetic patients were examined for natural killer cell activity using the K562 cell line as51Cr labeled targets. Mean natural killer cell cytotoxicity mediated by enriched non-T cells from patients (37 ± 4.0%) was lower (p < 0.03) than in controls (56 ± 3.7%). Specificity was evaluated by examining other patient subgroups. Mean non-T cell mediated natural killer cell activity in Type 2 (non-insulin-dependent) diabetic patients and Type 1 patients with long term disease was 65±54% and 62±4.8% respectively (p<0.003 vs new onset Type I patients). Longitudinal studies of new onset Type 1 patients during the remission (honeymoon) phase revealed persistently impaired natural killer cell activity in 3 of 4 patients. In 30 new onset and 11 remission Type 1 diabetic patients, mean non-T cell-mediated cytotoxicity was also measured using dispersed51Cr labeled islet target cells. Mean islet cytotoxicity mediated by cells from new onset patients was 34±2.4%, whereas in non-diabetic control subjects mean cytotoxicity was 25 ± 1.8% (p < 0.005). During remission, islet cytotoxicity remained at similar or elevated levels in most patients. In patients evaluated simultaneously for K562 and islet cell cytotoxicity, natural killer cell activity was decreased, whereas islet killing was increased. These results suggest a dichotomy in natural killer cell and islet killer cell activities in new onset Type 1 diabetes that could have an important role in the pathogenesis of Type diabetes.

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Natural killer cell and islet killer cell activities in Type 1 (insulin-dependent) diabetes

Diabetologia Natural killer cell and islet killer cell activities in Type 1 (insulin-dependent) diabetes K. Negishi 0 N. Waldeck 0 G. C h a n d y 0 B. Buckingham 0 A. K e r s h n a r 0 L. Fisher 0 S. G u p t a I 0 M. A. Charles ~ 0 0 Departments of 1Medicineand ZPhysiology,School of Medicine, Universityof California, Irvine, and Departments of Pediatrics, 3Children's Hospital of Orange County, Orange, and 4Cityof Hope Medical Center , Duarte, California , USA Summary. Peripheral blood mononuclear cells from 20 Type 1 (insulin-dependent) diabetic patients were examined for natural killer cell activity using the K562 cell line as 51Cr labeled targets. Mean natural killer cell cytotoxicity mediated by enriched non-T cells from patients (37 + 4.0%) was lower (p < 0.03) than in controls (56 _+3.7%). Specificity was evaluated by examining other patient subgroups. Mean non-T cell mediated natural killer cell activity in Type 2 (non-insulin-dependent) diabetic patients and Type 1 patients with long term disease was 65_+5.4% and 62_+4.8% respectively (p <0.003 vs new onset Type 1 patients). Longitudinal studies of new onset Type I patients during the remission (honeymoon) phase revealed persistently impaired natural killer cell activity in 3 of 4patients. In 30 new onset and 11 remission Type 1 diabetic Natural killer cells; islet cytotoxicity - I m m u n e mechanisms, genetic susceptibility and envir o n m e n t a l factors (e. g. viruses) are all implicated in the pathogenesis o f Type 1 (insulin-dependent) diabetes [ 1 ]. I m m u n e cytotoxic studies have focused u p o n antibodym e d i a t e d mechanisms o f islet destruction [ 2-4 ]. We have previously r e p o r t e d specifically increased cell-mediated cytotoxicity against islet target cells in Type 1 diabetes [ 4 ]. One potential cell-mediated killing mechanism for islet cell destruction Could be related to natural killer ( N K ) cells. N K cells are r e p o r t e d to have a significant role in resistance to viral infections [ 5 ]. It is docum e n t e d in animals [ 6 ] and suggested in h u m a n s [ 7 ] that viruses can cause pancreatic islet B cell destruction. Thus, it is conceivable that N K cells could be directly cytotoxic to islet cells, or that N K cell defects could result in susceptibility to viral infections, which in turn could play an i m p o r t a n t role in the pathogenesis o f Type 1 diabetes. Further, other diseases t h o u g h t to be a u t o i m m u n e in nature have b e e n associated with decreased N K cell populations or decreased K562 killing [ 8, 9 ]. We have recently r e p o r t e d preliminary results that Type 1 diabetes is associated with a quantitative and functional deficiency o f circulating N K cells using Leu 7 m o n o c l o n a l a n t i b o d y and K562 target cell cytotoxicity [ 10, 11 ]. Preliminary confirmation o f these data by Herald et al. [12] has also appeared. Since a dissociation between phenotypically defined N K cells and functionpatients, mean non-T cell-mediated cytotoxicity was also measured using dispersed 51Crlabeled islet target cells. Mean islet cytotoxicity mediated by cells from new onset patients was 34_+2.4%, whereas in non-diabetic control subjects mean cytotoxicity was 25 + 1.8% (p < 0.005). During remission, islet cytotoxicity remained at similar or elevated levels in most patients. In patients evaluated simultaneously for K562 and islet cell cytotoxicity, natural killer cell activity was decreased, whereas islet killing was increased. These results suggest a dichotomy in natural killer cell and islet killer cell activities in new onset Type I diabetes that could have an important role in the pathogenesis of Type diabetes. al abnormalities o f N K cells has b e e n described in other disorders [ 13 ], we c o m p a r e d the percent o f circulating H N K - 1 + m o n o n u c l e a r cells and N K cell cytotoxic function in Type 1 diabetes using target cells f r o m the K562 cell line. We also c o m p a r e d N K killing activity with cell-mediated islet cytotoxicity in Type 1 diabetic patients. Subjects and methods Subjects New onset TypeI diabetic patients were evaluated for islet and K562 cell cytotoxicity.Each patient and control subject understood and signed approved universityconsent forms prior to their participation in this project. Patient and control group age, sex and disease duration are described in Table1. The diagnostic criteria of Type1 and 2 diabetes have been previouslydescribed [ 14 ].The remission phase of diabetes was defined as an insulin requirement of < 0.5 U/kg per day, with normal fastingblood glucoselevelsof 1% or less urine testing for glucose occurring for longer than one week. Type2 diabetic control subjects all used insulin treatment. Normal subjects had no known immune abnormalities, normal fasting blood glucose levels and no familyhistoryof diabetes. For K562studies,the new onset Type1 patients and non-diabetic control group had similar numbers of males and females, since sex, but not age, is reported to be related to K562 cytotoxicity[ 15 ].Age~and sex-matchingfor islet cell cytotoxicityappears relativelyinsignificant,since diabetes-relatedcytotoxicityis elevated whether the patients are matched [ 4 ] or not (this study). Each subjectand patient was examined on one occasion,with the exception of patients studied longitudinally during the remission phase. Not all patients were examined for all parameters, but all studies performed are presented. Effector cell preparation Heparinized venous blood (200U/ml, preservative-free heparin; Gibco Laboratories, Grand Island, NY, USA) was used in all control subjects and diabetic patients. Enriched mononuclear cells were isolated using Ficoll-Hypaque (Pharmacia Fine Chemicals, Piscataway, NJ, USA) as described elsewhere [ 16 ]. Mononuclear cell preparations contained 60-80% T lymphocytes, 10% monocytes, 12-15% B lymphocytes and 5-8% null cells. Cells harvested from the interface were washed three times in Ca + + and Mg + + free Hanks' balanced salt solution (HBSS, Gibco). Non-T mononuclear cells were enriched by depletion of T cells using 1% 2-aminoethyl isothiouronium hydrobromide-treated sheep erythroeytes [ 17 ]. T lymphocyte rosettes were separated from non-T cells using Ficoll-Hypaque centrifugation. Non-T mononuclear cells obtained from the interface were washed three times with HBSS and resuspended in the appropriate culture medium. The non-T enriched mononuclear cell preparations contained 66% B cells, 20 25% monocytes, 15-20% null cells, and less than 5% T cells. Effector cell viability was greater than 95% by the trypan blue dye exclusion test. Target cell preparation The human myeloid K562 cell line was carried in culture as previously described [ 18 ]. K562 cells were grown in suspension culture using RPMI 1640 (Whittaker M.A. Bioproducts, Walkersville, MD, USA) and 10% fetal calf serum, 25 mol/1 Hepes buffer, penicillin and streptomycin. 2 x 10 6 K562 cells were labeled with 51Cr (200ul sodium chromate, 1 mCi/ml, New England Nuclear, Cambridge, MA, USA) at 37 °C for 60 min. After washing three times with culture medium, the labeled K562 cells were resuspended in the culture medium and adjusted to 1 x 105 cells/ml. Inbred Lewis rat islet cells were also used as target cells. Rat islets were isolated using collagenase, then were purified by centrifugation and visual selection as described previously [ 19 ]. Dispersed rat islet cells were prepared as described previously [ 3 ]. Purified intact islets were incubated at 37° for 1-2 days in Dulbecco's modified Eagles Medium (DMEM) and 10% fetal calf serum in 5% CO2 and 95% humidified atmosphrere. Individual intact islets were dispersed into a single cell suspension by incubation in 200 ug/ml DNAase (Sigma, St. Louis, MO, USA) in Swim's low calcium medium (Gibco) containing 1 mmol/1 EGTA (Sigma) and 1% bovine serum albumin (Miles Laboratories, Kankakee, IL, USA). The dispersed cells were washed, placed in 200 Ixl D M E M with 10% fetal calf serum and incubated with 51Cr (50 al sodium chromate, I mCi/ml, New England Nuclear, Cambridge, MA, USA) at 37° for 30 rain. After washing, 5aCr-labeled islet cells were used as target cells. Viability for both K562 and islet cells was greater than 90% by trypan blue exclusion. K562 N K cell assay Cytotoxicity against K562 target cells was performed in standard Vshaped microplates (Nunc, Kamstrups, Denmark) in a total volume of 200 !11as previously described [ 20 ]. 1 x 104 target cells were incubated with variable numbers of effector cells for 4 h at 37° under 5% CO2 and 95% humidified atmosphere. After incubation, 100 gl of supernatant was collected, the amount of 51Cr release was determined and the percent specific cytotoxicity from triplicate determinations was calculated as previously described [ 3 ] as follows: mean experimental cpm - mean spontaneous cpm x 100 mean maximum cpm - mean spontaneous cpm Mean spontaneous release determined from experiments (n = 20) after incubation of target cells was 9.4% (454 + 16 cpm), and maximal release using 20 gl of 10% Triton X-100 was 96% (4819 _+172 cpm). Islet cell cytotoxicity assay This assay was performed as described previously in detail [ 3, 4 ]. Triplicate 400 lxl polyethylene tubes contained MEM, 10% fetal calf serum, 10 4 5acr-labeled islet targe cells and variable quantities of effector cells. Incubations were performed for 8 h at 37° in 5% CO2 and 95% humidified atmosphere. After incubation, the tubes were mixed, centrifuged at 11600 x G for 2 min, and supematants and pellets were assessed for radioactivity. Spontaneous release was 21% (528+ 23 cpm) from tubes containing no added mononuclear cells. Maximal release using 1% Triton X-100 treated cells was 96% (2507 +_126 cpm). Specific cytotoxicity was calculated as described above for K562 target cells. Interassay variation was examined by repeated assays in one normal subject. Mean islet cytotoxicity was 25+3.8% at a 1:20 target :effector ratio (n =7). The islet assay was evaluated for islet target cell specificity as previously described [4], where rat conconavalin-A stimulated lymphoblast and rat macrophage target cells were not lyzed to the extent of islet cells. oo o o o co & o A ~ ,x [3 Monoclonal antibody (Leu 7+) defined cells The mononuclear cells were incubated with appropriate dilutions of fluorescein conjugated anti-Leu 7 monoclonal antibody (Bectin-Dickinson, Mountainside, Calif., USA) and Leu 7 + cells were enumerated with FACS analyzer (Bectin-Dickinson) as previously described [ 10 ]. Statistical analysis All data are presented as mean + SEM, and p values were determined using the Student's unpaired t-test unless otherwise stated. The Pearson correlation coefficient method was used. R e s u l t s K562 cytotoxicity Natural killer (NK) cell activity o f peripheral blood mononuclear cells from new onset Type 1 diabetic patients and non-diabetic control subjects was evaluated using K562 target cells. Mean cytotoxicity mediated b y enriched mononuclear cells at 1:10 and 1 : 50 target: effector ratios were lower in diabetic patients than in control subjects; these data have been previously published [ 11 ]. Mean enriched non-T cell killing o f K562 cells was lower in new onset Type 1 patients (1 : 10, 22 __+3.2%; 1 : 50, 37+_4.0%) than in control subjects (1: 10, 3 7 + 4 . 1 % (p < 0.03); 1 : 50, 56 _+3.7% (p < 0.03). To assess specificity o f N K cell activity to new onset Type 1 patients, N K activity was also measured in 2 other patient subtypes (Fig. 1). Mean N K activity in long-term Type 1 patients was 24 + 2.9% at a target: effector ratio o f 1 : 10 (p = NS vs new onset) and 62 _+4.8% at 1 : 50 (p < 0.03 vs new onset). Mean N K activity in Type 2 diabetic patients was 36_+5.4% at a target: effector ratio o f 1:10 (p <0.05 vs new onset) and 65 + 5.4% at 1 : 50 (p < 0.03 vs new onset). In new onset Type 1 patients who were longitudinally followed into the remission (honeymoon) phase (Fig. 1), N K activity remained impaired in 3 o f the 4 patients (p = NS vs new onset). M e a n levels o f circulating mononuclear cells expressing reactivity to Leu 7 were lower (9.0 + 0.9%) in new onset Type 1 patients than in non-diabetic controls (19 _+1.5%, p < 0.001, Fig. 2), which was also true for enriched non-T cells (14 + 2.2% vs 22 +- 1.8% in controls, p < 0.006, Fig. 3). Islet cytotoxicity We have previously reported in preliminary studies that islet cytotoxicity is increased using enriched mononuclear cells or enriched non-T cells from new onset Type I diabetic patients [ 4 ]. The current study confirms increased islet cytotoxicity b y non-T mononuclear cell preparations derived from 30 new onset Type 1 diabetic patients when compared to 29 non-diabetic control subjects (Fig. 4). As shown in Figure 5, islet cytotoxicity b y non-T cells during the remission phase o f diabetes in 11 patients was decreased toward normal values in 4, relatively unchanged in 4 and increased in 3 patients. I Jormal subjects I Type1 New onset Fig.4. Mean enriched non-T cell mediated islet cell cytotoxicity is shown for 29 non-diabetic control subjects (open circles) and 30 new onset Type I diabetic patients (closed circles). The bars indicate standard error of the mean at the target: effector ratios I : 5 (p = NS), 1 : 10 (p < 0.002) and 1 :20 (p < 0.005) o .o u~ 50 The mean remission level o f islet cytotoxicity was not significantly different from the mean new onset cytotoxicity levels. Comparisons o f cellular killing using K562 and islet target cells As described above for all o f the cross-sectionally evaluated new onset Type 1 patients and the non-diabetic control group, Type I patients show impaired killing against K562 cells and enhanced killing against islet target cells. To determine whether more direct relationships exist between K562 and islet cytotoxicity in Type 1 diabetes, seventeen new onset Type 1 patients were simultaneously evaluated for both K562 and islet non-T lymphocyte-mediated cellular cytotoxicity. Mean islet cytotoxicity at a target:effector ratio o f 1:20 was increased in these patients to 3 4 + 3 . 3 % compared to 25 _+1.8% in 29 control subjects (p < 0.01), whereas N K activity at a 1:50 target effector ratio was reduced to 37:3.0% versus 56_+ 3.7% in the non-diabetic controls (p < 0.002). To emphasize the discordant cytotoxicity of these two target cells, data from each new onset Type I patient is illustrated as the percent stimulation above the mean control level for islet target cells and the percent inhibition below the mean control level for K562 cells (Fig. 6). In these simultaneously studied new onset patients, a significant correlation was observed between o~ 180 ; / • / I 1 I [ I I0 20 30 40 50 Islet specific cyt0t0xicity(%) I 60 K562 and islet target cell killing (r--0.47, p=0.04, Fig. 7). Thus, patients with the higher K562 cytotoxicity also showed the higher islet cytotoxicity even though absolute levels of cytotoxicity for K562 cells was low and for islet cells was high. No significant correlation was observed between circulating Leu 7 + cells and islet killing (r = 0.03, p = NS, n = 7). Discussion This study indicates that N K cell activity against K562 target cells is decreased in new onset Type 1 diabetic patients, whereas islet cell killing is increased. These data are concordant with our previous preliminary results, in which we describe a decrease in the percent of monoclonal antibody-defined circulating N K cells, a decrease in N K functional activity and enhanced islet cell cytotoxicity in new onset Type 1 diabetes [4, 10, I1]. In this report we expand our preliminary results in evaluating functional N K activity in new onset Type 1 patients using K562 target cells. We also describe the specificity for impaired N K activity to new onset patients, and we compare K562 and islet cytotoxicity in simultaneously evaluated patients. It is known that enriched T cells also manifest cytotoxicity to K562 cells. Thus, the non-T enriched mononuclear cell populations used in this study do not define all K562 killing activity. Our observations of decreased circulating Leu 7 + N K cells are important since these data, combined with the killing data, imply a low level of N K cells using two different methods. Specificity of the N K activity impairment to new onset Type 1 patients is documented by the data in Figure 2, which shows that non-diabetic subjects, long term Type 1 and Type 2 diabetic patients manifest a similar range of N K activity. Disease activity in Type I patients was not associated with a change in the impairment of N K function in the 3 of 4 patients assessed by longitudinal evaluation of patients during the new onset and remission periods. Non-T cell-mediated islet cytotoxicity was also unrelated to clinical disease activity in 7 of the 11 patients longitudinally evaluated during the new onset and remission periods. Protein-calorie malnutrition, which is reversible after refeeding, has been associated with impaired N K function [ 21 ]. Thus, the potential for starvation related to new onset diabetes as a cause of impaired N K function seems unlikely, since the N K and islet functional defects were not altered toward normal levels during the remission phase when patients had normalized their metabolic control and gained weight. It appears that decreased K562 N K cell activity is a secondary manifestation of new onset Type 1 diabetes, since the defect is not demonstrable in chronic Type 1 patients. Studies of pre-clinical, islet cell antibody-positive subjects should provide more information regarding the onset of the N K impairment. If the decrease in K562 cell killing also occurs during the pre-clinical phase, this could provide an explanation for the viral infection susceptibility which is thought to have a major role in the pathogenesis o f Type I diabetes [I]. The decreased levels of circulating Leu 7 + N K cells and decreased K562 killing in diabetes could be due to several factors. One possibility is that interferon production is either deficient in Type 1 diabetes or that N K cell sensitivity to interferon is reduced. Interferon is k n o w n to e n h a n c e N K cell f u n c t i o n a n d r e c r u i t p r e - N K cells i n t o active N K cells [ 22 ]. A s e c o n d p o s s i b i l i t y is t h a t d e f i c i e n t I L - 2 p r o d u c t i o n , r e c e n t l y r e p o r t e d in T y p e 1 diabetes, c o u l d result in d e c r e a s e d i n t e r f e r o n p r o d u c t i o n a n d t h u s N K f u n c t i o n [ 22, 23 ]. R e c e n t l y I L - 3 h a s also b e e n s h o w n to e n h a n c e N K activity [ 24 ]. Finally, p r o s t a g l a n d i n s a n d i m m u n e c o m p l e x e s h a v e b e e n s h o w n to i n h i b i t N K cell f u n c t i o n [ 25 ]. T h e s e subs t a n c e s h a v e also b e e n r e p o r t e d t o be e l e v a t e d in the circ u l a t i o n in T y p e 1 d i a b e t e s [ 26, 27 ]. I n n e w o n s e t T y p e 1 diabetes, w h e n K 5 6 2 N K cell activity is low, islet c y t o t o x i c i t y is high, s u g g e s t i n g t h a t t h e e f f e c t o r cell specificity o r cell t y p e killing e a c h o f t h e s e t w o t a r g e t cells is different. T h e e f f e c t o r cell specificity h y p o t h e s i s (i. e. o n e cell t y p e killing b o t h t a r g e t cells) is s u p p o r t e d b y t h e c o r r e l a t i o n b e t w e e n K 5 6 2 a n d islet cell killing, w h e r e m o r e K 5 6 2 killing activity is ass o c i a t e d w i t h m o r e islet killing activity. T h u s , t h e elevated islet cell c y t o t o x i c i t y in T y p e 1 p a t i e n t s suggests specificity to islets, w h e r e a s t h e i m p a i r e d K 5 6 2 c y t o t o x i c i t y suggests d e c r e a s e d specificity. N e w o n s e t T y p e 1 d i a b e t e s is a s s o c i a t e d w i t h multiple cellular i m m u n e defects. 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K. Negishi, N. Waldeck, G. Chandy, B. Buckingham, A. Kershnar, L. Fisher, S. Gupta, M. A. Charles. Natural killer cell and islet killer cell activities in Type 1 (insulin-dependent) diabetes, Diabetologia, 1986, 352-357, DOI: 10.1007/BF00903343