Child's Nerv Syst (1992) 8:83-85
/liNg
9 Springer-Verlag 1992
Plasma/ -endorphin levels and natural killer cells in two cases of congenital indifference to pain Renato Bernardini 1, Alessandra Tint 2, Gaetano Mauceri 1, Maria Clorinda Mazzarino 3, Grazia Malaponte 3, Ausilia Nicosia 3, Enrico Parano 2, and Agata Fiumara 2 1 Laboratory of Neuroendocrinoimmunology, Institute of Pharmacology, z Pediatric Clinic, and 3 Department of Immunology, University of Catania School of Medicine, Viale Andrea Doria, 6, 1-95100 Catania, Italy Received March 4, 1991/Revised August 8, 1991
Abstract. We have investigated p l a s m a / % e n d o r p h i n (/~E), A C T H , and cortisol in two cases of congenital indifference to pain (CIP), a rare syndrome characterized by unresponsiveness to painful stimuli. As the two patients had frequent skin infections, we also studied lymphocyte response to mytogens in the absence or presence of/%E. In addition, we explored a series of lymphocyte m e m brane antigens related to different aspects of the immune response, such as CD3 +, CD4 +, CD8 +, B, N K Leu 7, Leu 9, and Leu 19, anti-interleukin-2 receptor (antiTAC). Plasma/~-E levels in the two patients were significantly higher than in controls, whereas plasma A C T H and cortisol levels were normal. Lymphocyte response to the mitogen phytohemagglutinin was normal. The expression of Leu 7, Leu 9, and Leu 19, three antigens related to natural killer cells, was decreased by a b o u t 50%. The results indicate that in the two cases of CIP studied, high plasma/~-E levels are associated with a reduction in the expression of natural killer cells. This suggests that the two p h e n o m e n a are specifically related to each other. These data represent further evidence of the possible pathophysiological relevance of the neuroendocrine-immune feedback. Key words: Natural killer cells - Opioids - Pain sensitivity
Congenital indifference to pain (CIP) is a rare syndrome transmitted by autosomal recessive inheritance and characterized by the absence of an appropriate response to noxious stimuli [1, 2, 8]. The patients are unable to feel painful sensations, although the other sensory functions appear intact. An opioid-dependent [6] and an opioid-independent [9] form of CIP have been described. We have measured/%endorphin (/%E), A C T H , and cortisol levels in two patients showing insensitivity to pain. It is k n o w n that A C T H and cortisol as well as /%E interfere with the immune response [5]. In particular, and Offprint requests to: R. Bernardini
other proopiomelanocortin (POMC) gene-related peptides have their receptors on a n u m b e r of immunocytes, including natural killer (NK) cells [5]. Because of the recurrence of skin infections in the two patients, we have investigated whether possible impairment of the immune response could be related to plasma/~-E levels in these opioid-dependent forms of congenital indifference to pain.
Patients and methods Patients The patients were two brothers, born to healthy consanguineous parents. They did not respond to painful stimuli, and they had inflicted severe injury upon themselves by biting their own tongues and hands. At the time of diagnosis they were 8 and 14 years old respectively. Both brothers had a good general appearance, with weight and height in the 50th percentile. The head circumference was normal in the older brother but in the third percentile in the younger [10]. The patients had thick, inelastic skin; wounds caused by inadvertent self-mutilation, spontaneous ulcers, and shortness of the distal phalanxes. The elder brother's right lower limb had been replaced by a prosthesis because of a fracture complicated by gangrenous infection. Moderate-to-severe mental retardation was present. The patients did not respond to painful stimuli, whereas tactile sensations were preserved. Testing for touch, position, vibration, and temperature yielded inconsistent results because of the mental retardation. Deep tendon reflexes were normal [10]. The patients suffered from frequent skin infections.
Lymphocyte cultures Twenty to twenty-five milliliters of blood were diluted 1 : 1 with Ca ++-Mg + § phosphate-buffered saline (PBS, pH 7.4; Gibco, Long Island, NY), and subsequently stratified onto a 50% (v/v) Ficoll-Hypaque gradient (Pharmacia, Piscataway, N J). Samples were then spun at 1800 rpm for 30 min at room temperature. The resulting buffy coat of peripheral blood mononucleates (PBM) was separated and washed twice in PBS. The pellet was resuspended in 3 ml 10% fetal calf serum RPMI 1640 with •-glutamine (both from Gibco). An aliquot of 10 gl was diluted in 80 gl PBS, and ceils were
84 counted in a chamber, adding 10 gl trypan blue. The volume of cell solution was then adjusted, and cells were plated in a 96-well plate (Costar, Cambridge, MA), at a density of 2 x 10s/100 gl per well. Phytohemagglutinin (PHA, 100 gl, Sigma Chemical Co., St. Louis, MO) was added at concentrations of 20, 10, 5, 2.5, 1.2, and 0.6 gg/ ml. In other experiments, fl-E (1 p M to 1 gM, Sigma) was added to cultures, to test the responsiveness of patients' lymphocytes to it. Plates were incubated for 72 h in 5% CO z atmosphere, at 37~ Then cells were incubated for 6 h with 1 gCi/well tritiated thymidine (New England Nuclear, Florence, Italy; volume: 20 pl/well) and harvested onto a fiberglass filter (Skatron, Norway). Heat-dried filters were placed in plastic tubes and, after the addition of 3.5 ml scintillating solution (Instagel, Packard, The Netherlands), radioactivity was counted for 2 min in a fl-counter. After 72 h incubation with 5 gg/ml PHA, a 20-gl aliquot of anti-IL-2 receptor fluorescent antibody (anti-TAC; Becton-Dickinson, San Jose, Calif.) was added to 1 ml cells (1 x 106) and the percentage of fluorescent cells counted by means of fluorescence microscope. Other aliquots of cells were used immediately after separation of the buffy coat and incubated with the proper monoclonal antibody versus surface antigens for CD3 +, CD4 § CD8 +, B, and N K Leu 7, 9, and 19 (Boehringer, Mannheim, FRG).
= 100 E
CIP
ml]]]]] Controls
T ul 7 5 >
50-
o
E o r
25E {#1 a_
0
13-endorphin
ACTH
Fig. 1. Plasma A C T H and fl-endorphin levels in two patients with congenital indifference to pain (CIP). * P < 0.01 (Student's t test for each hormone measured); control subjects n = 10
150 o=
Plasma hormone assays Plasma fl-E, ACTH, and cortisol levels were measured by radioimmunoassay. Intra- and interassay coefficients of variation were 7% and 11% respectively for fl-E, 9% and 6% for ACTH, and 5% and 14% for cortisol.
o s i_ o
u c_
120 90
6O E o
27 Results
3O 0
Plasma fl-endorphin, ACTH, and cortisol
No differences in the levels of plasma ACTH (Fig. 1) and cortisol (data not shown) were seen between the patients and the controls. However, we found significantly higher levels of plasma fl-E in the patients than in the control subjects (P
/liNg
9 Springer-Verlag 1992
Plasma/ -endorphin levels and natural killer cells in two cases of congenital indifference to pain Renato Bernardini 1, Alessandra Tint 2, Gaetano Mauceri 1, Maria Clorinda Mazzarino 3, Grazia Malaponte 3, Ausilia Nicosia 3, Enrico Parano 2, and Agata Fiumara 2 1 Laboratory of Neuroendocrinoimmunology, Institute of Pharmacology, z Pediatric Clinic, and 3 Department of Immunology, University of Catania School of Medicine, Viale Andrea Doria, 6, 1-95100 Catania, Italy Received March 4, 1991/Revised August 8, 1991
Abstract. We have investigated p l a s m a / % e n d o r p h i n (/~E), A C T H , and cortisol in two cases of congenital indifference to pain (CIP), a rare syndrome characterized by unresponsiveness to painful stimuli. As the two patients had frequent skin infections, we also studied lymphocyte response to mytogens in the absence or presence of/%E. In addition, we explored a series of lymphocyte m e m brane antigens related to different aspects of the immune response, such as CD3 +, CD4 +, CD8 +, B, N K Leu 7, Leu 9, and Leu 19, anti-interleukin-2 receptor (antiTAC). Plasma/~-E levels in the two patients were significantly higher than in controls, whereas plasma A C T H and cortisol levels were normal. Lymphocyte response to the mitogen phytohemagglutinin was normal. The expression of Leu 7, Leu 9, and Leu 19, three antigens related to natural killer cells, was decreased by a b o u t 50%. The results indicate that in the two cases of CIP studied, high plasma/~-E levels are associated with a reduction in the expression of natural killer cells. This suggests that the two p h e n o m e n a are specifically related to each other. These data represent further evidence of the possible pathophysiological relevance of the neuroendocrine-immune feedback. Key words: Natural killer cells - Opioids - Pain sensitivity
Congenital indifference to pain (CIP) is a rare syndrome transmitted by autosomal recessive inheritance and characterized by the absence of an appropriate response to noxious stimuli [1, 2, 8]. The patients are unable to feel painful sensations, although the other sensory functions appear intact. An opioid-dependent [6] and an opioid-independent [9] form of CIP have been described. We have measured/%endorphin (/%E), A C T H , and cortisol levels in two patients showing insensitivity to pain. It is k n o w n that A C T H and cortisol as well as /%E interfere with the immune response [5]. In particular, and Offprint requests to: R. Bernardini
other proopiomelanocortin (POMC) gene-related peptides have their receptors on a n u m b e r of immunocytes, including natural killer (NK) cells [5]. Because of the recurrence of skin infections in the two patients, we have investigated whether possible impairment of the immune response could be related to plasma/~-E levels in these opioid-dependent forms of congenital indifference to pain.
Patients and methods Patients The patients were two brothers, born to healthy consanguineous parents. They did not respond to painful stimuli, and they had inflicted severe injury upon themselves by biting their own tongues and hands. At the time of diagnosis they were 8 and 14 years old respectively. Both brothers had a good general appearance, with weight and height in the 50th percentile. The head circumference was normal in the older brother but in the third percentile in the younger [10]. The patients had thick, inelastic skin; wounds caused by inadvertent self-mutilation, spontaneous ulcers, and shortness of the distal phalanxes. The elder brother's right lower limb had been replaced by a prosthesis because of a fracture complicated by gangrenous infection. Moderate-to-severe mental retardation was present. The patients did not respond to painful stimuli, whereas tactile sensations were preserved. Testing for touch, position, vibration, and temperature yielded inconsistent results because of the mental retardation. Deep tendon reflexes were normal [10]. The patients suffered from frequent skin infections.
Lymphocyte cultures Twenty to twenty-five milliliters of blood were diluted 1 : 1 with Ca ++-Mg + § phosphate-buffered saline (PBS, pH 7.4; Gibco, Long Island, NY), and subsequently stratified onto a 50% (v/v) Ficoll-Hypaque gradient (Pharmacia, Piscataway, N J). Samples were then spun at 1800 rpm for 30 min at room temperature. The resulting buffy coat of peripheral blood mononucleates (PBM) was separated and washed twice in PBS. The pellet was resuspended in 3 ml 10% fetal calf serum RPMI 1640 with •-glutamine (both from Gibco). An aliquot of 10 gl was diluted in 80 gl PBS, and ceils were
84 counted in a chamber, adding 10 gl trypan blue. The volume of cell solution was then adjusted, and cells were plated in a 96-well plate (Costar, Cambridge, MA), at a density of 2 x 10s/100 gl per well. Phytohemagglutinin (PHA, 100 gl, Sigma Chemical Co., St. Louis, MO) was added at concentrations of 20, 10, 5, 2.5, 1.2, and 0.6 gg/ ml. In other experiments, fl-E (1 p M to 1 gM, Sigma) was added to cultures, to test the responsiveness of patients' lymphocytes to it. Plates were incubated for 72 h in 5% CO z atmosphere, at 37~ Then cells were incubated for 6 h with 1 gCi/well tritiated thymidine (New England Nuclear, Florence, Italy; volume: 20 pl/well) and harvested onto a fiberglass filter (Skatron, Norway). Heat-dried filters were placed in plastic tubes and, after the addition of 3.5 ml scintillating solution (Instagel, Packard, The Netherlands), radioactivity was counted for 2 min in a fl-counter. After 72 h incubation with 5 gg/ml PHA, a 20-gl aliquot of anti-IL-2 receptor fluorescent antibody (anti-TAC; Becton-Dickinson, San Jose, Calif.) was added to 1 ml cells (1 x 106) and the percentage of fluorescent cells counted by means of fluorescence microscope. Other aliquots of cells were used immediately after separation of the buffy coat and incubated with the proper monoclonal antibody versus surface antigens for CD3 +, CD4 § CD8 +, B, and N K Leu 7, 9, and 19 (Boehringer, Mannheim, FRG).
= 100 E
CIP
ml]]]]] Controls
T ul 7 5 >
50-
o
E o r
25E {#1 a_
0
13-endorphin
ACTH
Fig. 1. Plasma A C T H and fl-endorphin levels in two patients with congenital indifference to pain (CIP). * P < 0.01 (Student's t test for each hormone measured); control subjects n = 10
150 o=
Plasma hormone assays Plasma fl-E, ACTH, and cortisol levels were measured by radioimmunoassay. Intra- and interassay coefficients of variation were 7% and 11% respectively for fl-E, 9% and 6% for ACTH, and 5% and 14% for cortisol.
o s i_ o
u c_
120 90
6O E o
27 Results
3O 0
Plasma fl-endorphin, ACTH, and cortisol
No differences in the levels of plasma ACTH (Fig. 1) and cortisol (data not shown) were seen between the patients and the controls. However, we found significantly higher levels of plasma fl-E in the patients than in the control subjects (P
