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Deficiency of monoclonal antibody (Leu 7) defined NK cells in newly diagnosed insulin-dependent diabetes mellitus
Immunolog~ Letter~. ~ 11984) 89 9[ Ir Ise~ier Imlet 483
DEFICIENCY OF MONOCLONAL A N T I B O D Y ( L e u 7) D E F I N E D N K C E L L S IN NEWLY DIAGNOSED INSULIN-DEPENDENT DIABETES MELLITUS K. G. CHANDY I, M. A. CHARLES-', B. BUCKINGHAM 3, N. WALDECK 3, A. KERSHNAR -~ and S. GUPTA ] I Dtvi~/ons 611"Basl~ and Climcal Immunolog.i, and 2Endocrinologr, Department of Me~hctne. I.'m~erstt.~ of Cal!/orma, Irvine. and ~Chiklren's Hospital of Orange Countl. Orange, C.-I, U.S.A. (Recei,,ed 3 Januar 3 1984) (Modified ~ersion received 20 March 1984) (Accepted 27 March 1984)
viral-induced islet cell injury, contributing to the pathogenesis of IDDM.
I. Summary
Peripheral blood from II newly diagnosed patients with insulin-dependent diabetes mellitus (IDDM) was studied for the proportion of monoclonal antibody (HNK I, Leu 7) defined natural killer (NK) cells using a fluorescence-activated cell sorter analyzer. The proportion of Leu 7 + cells in patients with IDDM (7.0 ± 4.0) was significantly (P < 0.001 ) lower than in simultaneously studied healthy controls ( 16.8 + 7.0). A 2-yr-old boy with recent onset IDDM had a deficiency of Leu 7 + NK cells (6. I%), while his healthy identical twin had normal proportions of Leu 7 + cells (22.2%), when compared to a simultaneously studied healthy control. Two patients reexamined in remission and one other studied in remission alone, showed deficiency of Leu 7 + NK cells. This study demonstrates a quantitative deficiency of monoclonal antibody (Leu 7 +) defined NK cells in newly diagnosed patients with IDDM that persists during remission of the disease and therefore appears to be independent of metabolic abnormality. The deficiency of NK cells may predispose genetically susceptible individuals to
,4ddre~s correspondence to: K. George Chandy, M.D., Ph.D,. Division of Basic and Clinical Immunology, Med. ScL I. Room C-264, University of California. Irvme, CA 92717, U.S.A.
2. Introduction
Immunological, environmental and genetic factors have been implicated in the pathogenesis of insulindependent diabetes mellitus (IDDM). Among the environmental factors, virus(es) appear to play an important role in the etiopathogenesis of IDDM [I 2]. Induction of IDDM in mice by the M variant of encephalomyocarditis virus is consistent with this hypothesis [3]. Natural killer (NK) cells are important in resistance against virus infections, rapidly lysing (within hours) vitally infected cells [4]. A recently developed monoclonal antibody, HNK-I (Leu 7), has been reported to be specific for human NK cells, reacting with almost all (92%) large granular cells [5]. In the present study we have examined the proportion of Leu 7 + cells in the peripheral blood of patients with newly diagnosed IDDM and healthy controls, to determine whether NK cells play a role in the pathogenesis of IDDM.
3. Methods 3. I. Patients
Key ~,ords." msulin-dependent diabetes mellitus cells
natural kdler
monoclonal ant~bod.~ I Leu 7)
0165-2478
84
$3 00 © Elsevier Science Publishers B V.
Eleven patients with recent onset IDDM, I patient with IDDM in remission alone and 32 healthy 89
Table I Characteristics of pauents ,a Jth nev. I} diagnosed I D D M
Patients
.Age ~.~rl ~ex
I
~ F
2 3 4
26 M 8 M 16 M
5 6
10 M 7 M 9.5 F 7 M 12 M 33 M
8 9 l0 II 12
13
9 F II F
2 M
Duration Ida,<
HbAIC
2 Remi~,ton '
9 l0
ND+ 05) ND II.0 13.0 8 3 ND+ "2 ND+ ~." ND+ ND+
IO
ND+
Remission "
8 b ND
20 II
8 RemMion " 14 2
Defined as requirements of insulin ."0.3 U per kg ,at per d a y a n d n o r m a l fasting blood glucose. N D + = Not d o n e The ages ol health} controls '.aned front 20 t o 4 2 }r.
controls ~ere included in this stud}'. Clinical and labo r a t o r ' data are shown in Table I. in addition, a 2yr-old child ~ith recent onset I D D M and his health}' identical twin were examined.
Dickinson, Mountainside. CA). Cells from pauents and health}' controls were examined simultaneousl\. 3.4. Statisth'al tests Differences in the proportion of Leu 7 + cells between patients and controls were assessed by the significance test for a difference in mean between two independent sample populations [6].
4. Results and Discussion NK cells play an important role in resistance against viral infections. Deficienc.~ of NK cells could result in the persistence of x irus resulting in its cx topathic effect on B cells of islets, manifesting as I D D M . On the other hand, ~hile attempting to combat xiral infection, NK cells could directly damage islet cells. In the present studx we estimated the numbers of Leu 7 + NK cells to ascertain ~hether NK cells are invol~ed in the etiopathogenesis of IDDM. Results are shown in Fig. I. The absolute lymphocyte counts in the patients and controls ~ere comparable and therefore data are presented as per3"2'
3.2. Isolation o f mononuclear cells Mononuclear cells from fresh heparinized (20 U ml) peripheral venous blood were separated on FicolI-Hypaque density gradient, ~ashed 3 times with Hanks" balanced salt solution (HBSS, Irvine Scientific Co., Irxine) and resuspended in HBSS at a concentration of 10 × 106 cells' ml. Viability was >98% as determined by trypan blue dye exclusion. 3.3..4 nah'sis o,f N K cells b.l' Leu 7 monoclonal antibody Cells were aliquoted into I ml capacity microcentrifuge tubes (I × 10°~tube), washed ~ith phosphate buffered saline (PBS. pH 7.2) and resuspended in 50 ul of a 1:20 dilution of fluorescein conjugated Leu 7 (Becton-Dickinson, Sunnyvale, CA) monoclonal antibody in PBS. The mi,qure ~as incubated on ice for 30 min, ~ashed 3 times in PBS and resuspended in 0.5 ml PBS. The proportion of Leu 7 + cells was assessed by a fluorescence-activated celt sorter analyzer ( F A C S Analyzer. Becton90
J
..r.
L~
2'3
W-
z ~2
i
z .2,
I"7.,' .%
&
t}_
rJewl¢
ff~e rr,,~,,',r,
17.,,rJg nOSed
,~o r i T P,_SL':o
IDDM Fig. I. Prc, p o r h o n o f v, lth I D D M
HNK-I'
I Leu " l cell., in pat~enl,
and health) control~
Mean_+SD"
control,, - Ib ~ + 7.O: patJent, - ",.O -¢-4 O: [ ] = Health', t ~ m ~I 2-.~r-c, ld bo', 'alth I D D M :
• -2-yr-old
bo~, ~'.ith I D D M
cent of mononuclear cells. Patients with newly diagnosed I D D M had significantly looser ( P < 0.001 ) lexels of Leu 7 + cells (mean % +_ SD; 7.0 ___4.0) when compared with controls (mean % _+ SD; 16.8 _+ 7.0). A 2-yr-old boy with recent onset I D D M had a deficienc3 of Leu 7 + cells 16.1%) while his healthy twin had normal proportions (22.2e~) when compared to a simultaneously studied healthy control. Ts~o patients reexamined in remission and one studied in remission alone also had low lexels of NK cells. The data suggest that decreased numbers of Leu 7 + NK cells in recent onset I D D M could be a primary abnormalit3, independent of metabolic disturbances. Our preliminar~ studies (data not shown) indicate that NK actixity against K-562 cells as targets, is also deficient in newl.~ diagnosed I D D M . The deficiency of Leu 7 + cells could predispose a genetically susceptible individual to viral-induced islet cell injur3. Recentl.~, another monoclonal antibody, Leu II (NKP-15). that defines a surface antigen on NK cells has been described [7]. This antibody also appears to react with mature neutrophils and basophils [7]. Lanier and Loken [8] demonstrated a greater heterogeneit.v among cells defined with Leu 7 and Leu I I monoclonal antibodies. Using three colour immunofluorescence they demonstrated that Leu 7 antibod3 in addition to reacting with Leu I + and Leu 4 ÷ cells [7.9] also reacts with a subset of Leu 2 +, Leu 3 + and Leu I I + cells. Leu I I antibody does not react ~ith either Leu I ÷ or Leu 4 ÷ T cells; howe~,er. approximately I-2e~ Leu 2 + cells reacted with Leu II antibody. Lanier and Loken [8] have sho~ n earlier that Leu I I ÷, 7 cells are most potent in NK, fol-
lowed by keu 7 +, I I and cells with Leu 7 +, I I ÷ lack NK functions completely. Therefore studies are required using t ~ o fluorochromes and both Leu II and Leu 7 monoclonal antibodies to define these subsets of NK cells.
Acknowledgements This x~ork ~as supported by the American Diabetes Association, Southern California Affiliate, Inc. Fellowship Ax~ard (K.G.C.) and by the American Diabetes Association, Southern California Affiliate. Inc. Research Grant A ~ a r d (S.G. and M.A.C.).
References [I] Yoon. J.. Austin. M.. Onodera. "I[. and Notkm.,, ~. (19'~9) N Eng] J. Med. 300. 11"7'3 11"79 [2] H u l ~ r . S . A . and MacPherson. B R.(19831m:Immunolog3 of Chmcal and ExperlmentaIDiabete_,,fS Gupta. E d ) i n press. Plenum Press. Ne',~ ~' ork. [3] Cralghead. J E. and MacLane. M F. B. 1196SI Science tO2, q13 q14. [4] Santoh. D . Peruss]a. B. and TrmchtarL O (]qgO) in: Natural Cell Mediated Immunit.~ Against Tumors (R. B. Herberman. Ed I p. 1171. ,~cademlc Press. Ne'a York [5] Abo. T and Balch. C M (1980) J Immunol 12". 1024 1029 [6] Bahn. A. K. 11972) in: Basic Medical Statisttcs. pp. 137 139. Grune and Stratton. Ne,.~ ~ ork. [7] Lanier. L. L..Le.A M.Ph]lhps, J. H..~,~arner. N L and Babcock. G F (19831J. Immunol. 131. 1789. [8] Lanier. L. L. and Loken. M.R.(19841J Immunol 132. 151 [9] Abo, T..Cooper. M D. and Balch. C M. IIqS21J Immunol. 129. 1"7,52.
91
DEFICIENCY OF MONOCLONAL A N T I B O D Y ( L e u 7) D E F I N E D N K C E L L S IN NEWLY DIAGNOSED INSULIN-DEPENDENT DIABETES MELLITUS K. G. CHANDY I, M. A. CHARLES-', B. BUCKINGHAM 3, N. WALDECK 3, A. KERSHNAR -~ and S. GUPTA ] I Dtvi~/ons 611"Basl~ and Climcal Immunolog.i, and 2Endocrinologr, Department of Me~hctne. I.'m~erstt.~ of Cal!/orma, Irvine. and ~Chiklren's Hospital of Orange Countl. Orange, C.-I, U.S.A. (Recei,,ed 3 Januar 3 1984) (Modified ~ersion received 20 March 1984) (Accepted 27 March 1984)
viral-induced islet cell injury, contributing to the pathogenesis of IDDM.
I. Summary
Peripheral blood from II newly diagnosed patients with insulin-dependent diabetes mellitus (IDDM) was studied for the proportion of monoclonal antibody (HNK I, Leu 7) defined natural killer (NK) cells using a fluorescence-activated cell sorter analyzer. The proportion of Leu 7 + cells in patients with IDDM (7.0 ± 4.0) was significantly (P < 0.001 ) lower than in simultaneously studied healthy controls ( 16.8 + 7.0). A 2-yr-old boy with recent onset IDDM had a deficiency of Leu 7 + NK cells (6. I%), while his healthy identical twin had normal proportions of Leu 7 + cells (22.2%), when compared to a simultaneously studied healthy control. Two patients reexamined in remission and one other studied in remission alone, showed deficiency of Leu 7 + NK cells. This study demonstrates a quantitative deficiency of monoclonal antibody (Leu 7 +) defined NK cells in newly diagnosed patients with IDDM that persists during remission of the disease and therefore appears to be independent of metabolic abnormality. The deficiency of NK cells may predispose genetically susceptible individuals to
,4ddre~s correspondence to: K. George Chandy, M.D., Ph.D,. Division of Basic and Clinical Immunology, Med. ScL I. Room C-264, University of California. Irvme, CA 92717, U.S.A.
2. Introduction
Immunological, environmental and genetic factors have been implicated in the pathogenesis of insulindependent diabetes mellitus (IDDM). Among the environmental factors, virus(es) appear to play an important role in the etiopathogenesis of IDDM [I 2]. Induction of IDDM in mice by the M variant of encephalomyocarditis virus is consistent with this hypothesis [3]. Natural killer (NK) cells are important in resistance against virus infections, rapidly lysing (within hours) vitally infected cells [4]. A recently developed monoclonal antibody, HNK-I (Leu 7), has been reported to be specific for human NK cells, reacting with almost all (92%) large granular cells [5]. In the present study we have examined the proportion of Leu 7 + cells in the peripheral blood of patients with newly diagnosed IDDM and healthy controls, to determine whether NK cells play a role in the pathogenesis of IDDM.
3. Methods 3. I. Patients
Key ~,ords." msulin-dependent diabetes mellitus cells
natural kdler
monoclonal ant~bod.~ I Leu 7)
0165-2478
84
$3 00 © Elsevier Science Publishers B V.
Eleven patients with recent onset IDDM, I patient with IDDM in remission alone and 32 healthy 89
Table I Characteristics of pauents ,a Jth nev. I} diagnosed I D D M
Patients
.Age ~.~rl ~ex
I
~ F
2 3 4
26 M 8 M 16 M
5 6
10 M 7 M 9.5 F 7 M 12 M 33 M
8 9 l0 II 12
13
9 F II F
2 M
Duration Ida,<
HbAIC
2 Remi~,ton '
9 l0
ND+ 05) ND II.0 13.0 8 3 ND+ "2 ND+ ~." ND+ ND+
IO
ND+
Remission "
8 b ND
20 II
8 RemMion " 14 2
Defined as requirements of insulin ."0.3 U per kg ,at per d a y a n d n o r m a l fasting blood glucose. N D + = Not d o n e The ages ol health} controls '.aned front 20 t o 4 2 }r.
controls ~ere included in this stud}'. Clinical and labo r a t o r ' data are shown in Table I. in addition, a 2yr-old child ~ith recent onset I D D M and his health}' identical twin were examined.
Dickinson, Mountainside. CA). Cells from pauents and health}' controls were examined simultaneousl\. 3.4. Statisth'al tests Differences in the proportion of Leu 7 + cells between patients and controls were assessed by the significance test for a difference in mean between two independent sample populations [6].
4. Results and Discussion NK cells play an important role in resistance against viral infections. Deficienc.~ of NK cells could result in the persistence of x irus resulting in its cx topathic effect on B cells of islets, manifesting as I D D M . On the other hand, ~hile attempting to combat xiral infection, NK cells could directly damage islet cells. In the present studx we estimated the numbers of Leu 7 + NK cells to ascertain ~hether NK cells are invol~ed in the etiopathogenesis of IDDM. Results are shown in Fig. I. The absolute lymphocyte counts in the patients and controls ~ere comparable and therefore data are presented as per3"2'
3.2. Isolation o f mononuclear cells Mononuclear cells from fresh heparinized (20 U ml) peripheral venous blood were separated on FicolI-Hypaque density gradient, ~ashed 3 times with Hanks" balanced salt solution (HBSS, Irvine Scientific Co., Irxine) and resuspended in HBSS at a concentration of 10 × 106 cells' ml. Viability was >98% as determined by trypan blue dye exclusion. 3.3..4 nah'sis o,f N K cells b.l' Leu 7 monoclonal antibody Cells were aliquoted into I ml capacity microcentrifuge tubes (I × 10°~tube), washed ~ith phosphate buffered saline (PBS. pH 7.2) and resuspended in 50 ul of a 1:20 dilution of fluorescein conjugated Leu 7 (Becton-Dickinson, Sunnyvale, CA) monoclonal antibody in PBS. The mi,qure ~as incubated on ice for 30 min, ~ashed 3 times in PBS and resuspended in 0.5 ml PBS. The proportion of Leu 7 + cells was assessed by a fluorescence-activated celt sorter analyzer ( F A C S Analyzer. Becton90
J
..r.
L~
2'3
W-
z ~2
i
z .2,
I"7.,' .%
&
t}_
rJewl¢
ff~e rr,,~,,',r,
17.,,rJg nOSed
,~o r i T P,_SL':o
IDDM Fig. I. Prc, p o r h o n o f v, lth I D D M
HNK-I'
I Leu " l cell., in pat~enl,
and health) control~
Mean_+SD"
control,, - Ib ~ + 7.O: patJent, - ",.O -¢-4 O: [ ] = Health', t ~ m ~I 2-.~r-c, ld bo', 'alth I D D M :
• -2-yr-old
bo~, ~'.ith I D D M
cent of mononuclear cells. Patients with newly diagnosed I D D M had significantly looser ( P < 0.001 ) lexels of Leu 7 + cells (mean % +_ SD; 7.0 ___4.0) when compared with controls (mean % _+ SD; 16.8 _+ 7.0). A 2-yr-old boy with recent onset I D D M had a deficienc3 of Leu 7 + cells 16.1%) while his healthy twin had normal proportions (22.2e~) when compared to a simultaneously studied healthy control. Ts~o patients reexamined in remission and one studied in remission alone also had low lexels of NK cells. The data suggest that decreased numbers of Leu 7 + NK cells in recent onset I D D M could be a primary abnormalit3, independent of metabolic disturbances. Our preliminar~ studies (data not shown) indicate that NK actixity against K-562 cells as targets, is also deficient in newl.~ diagnosed I D D M . The deficiency of Leu 7 + cells could predispose a genetically susceptible individual to viral-induced islet cell injur3. Recentl.~, another monoclonal antibody, Leu II (NKP-15). that defines a surface antigen on NK cells has been described [7]. This antibody also appears to react with mature neutrophils and basophils [7]. Lanier and Loken [8] demonstrated a greater heterogeneit.v among cells defined with Leu 7 and Leu I I monoclonal antibodies. Using three colour immunofluorescence they demonstrated that Leu 7 antibod3 in addition to reacting with Leu I + and Leu 4 ÷ cells [7.9] also reacts with a subset of Leu 2 +, Leu 3 + and Leu I I + cells. Leu I I antibody does not react ~ith either Leu I ÷ or Leu 4 ÷ T cells; howe~,er. approximately I-2e~ Leu 2 + cells reacted with Leu II antibody. Lanier and Loken [8] have sho~ n earlier that Leu I I ÷, 7 cells are most potent in NK, fol-
lowed by keu 7 +, I I and cells with Leu 7 +, I I ÷ lack NK functions completely. Therefore studies are required using t ~ o fluorochromes and both Leu II and Leu 7 monoclonal antibodies to define these subsets of NK cells.
Acknowledgements This x~ork ~as supported by the American Diabetes Association, Southern California Affiliate, Inc. Fellowship Ax~ard (K.G.C.) and by the American Diabetes Association, Southern California Affiliate. Inc. Research Grant A ~ a r d (S.G. and M.A.C.).
References [I] Yoon. J.. Austin. M.. Onodera. "I[. and Notkm.,, ~. (19'~9) N Eng] J. Med. 300. 11"7'3 11"79 [2] H u l ~ r . S . A . and MacPherson. B R.(19831m:Immunolog3 of Chmcal and ExperlmentaIDiabete_,,fS Gupta. E d ) i n press. Plenum Press. Ne',~ ~' ork. [3] Cralghead. J E. and MacLane. M F. B. 1196SI Science tO2, q13 q14. [4] Santoh. D . Peruss]a. B. and TrmchtarL O (]qgO) in: Natural Cell Mediated Immunit.~ Against Tumors (R. B. Herberman. Ed I p. 1171. ,~cademlc Press. Ne'a York [5] Abo. T and Balch. C M (1980) J Immunol 12". 1024 1029 [6] Bahn. A. K. 11972) in: Basic Medical Statisttcs. pp. 137 139. Grune and Stratton. Ne,.~ ~ ork. [7] Lanier. L. L..Le.A M.Ph]lhps, J. H..~,~arner. N L and Babcock. G F (19831J. Immunol. 131. 1789. [8] Lanier. L. L. and Loken. M.R.(19841J Immunol 132. 151 [9] Abo, T..Cooper. M D. and Balch. C M. IIqS21J Immunol. 129. 1"7,52.
91