Immunological abnormality in patients with lysinuric protein intolerance

JOURNAL

OF THE

NEUROLOGICAL SCIENCES Journal of the Neurological Sciences 134 (1995) 178-182

ELSEVIER

Immunological

abnormality in patients with lysinuric protein intolerance

Yoshihiro Yoshida a7*, Koichi Machigashira b, Masahito Suehara b, Hitoshi Arimura ‘, Takashi Moritoyo b, Keiji Nagamatsu ‘, Mitsuhiro Osame b a School ofAllied ’ 3rd Department of Internal

Medical Sciences, Kagoshima Uniuersity, 8-35-1, Sakuragaoka, Kagoshima Medicine, Faculty of Medicine, Kagoshima Uniuersity 8-35-1, Sakuragaoka, ’ Ohita Prefectural Hospital, 476, Bunyo, Ohita 870, Japan

890, Japan Kagoshima

890, Japan

Received 23 February 1995; revised 26 June 1995; accepted 19 July 1995

Abstract Lysinuric protein intolerance (LPI) is a rare hereditary disorder manifesting hyperammonemia induced by low levels of basic amino acids, these low levels being due to the impaired transport of these acids in the intestinal mucosa and the renal tubules. Low serum arginine levels and probably the consequently low in vivo levels of nitric oxide (NO), which against acts as a physiological and immunological mediator/modulator, are thought to influence the immunological status in patients with LPI. Accordingly, this study was conducted to. We found that patients with LPI had leukocytopenia, high serum IgG levels, a high ratio of CD44B4-positive lymphocytes (helper inducer) to CD42H4-positive lymphocytes (suppressor inducer), low levels of leukocyte phagocytic, cytotoxic, and natural killer cell activity, and increased spontaneous proliferation of lymphocytes. These results were probably the consequence of persistent low NO levels in vivo. Keywords:

Lysinuric protein intolerance; Macrophage; Lymphocyte; Subpopulation; IgC; Natural killer cell

1. Introduction

intestinal flora, since germ-free rats have also been shown to excrete

Lysinuric protein intolerance (LPI) is a rare hereditary disorder of the urea cycle. The impaired transport of the dibasic amino acids, arginine, ornithine, and lysine, in the intestinal mucosa and renal tubules leads to a decreasein plasma levels and an increasein urinary excretion of these amino acids (Perheentupaand Visakorpi, 1965; Kekomaki et al., 1967; Simell et al., 1975). The consequentornithine deficiency causes episodic hyperammonemia and erotic aciduria (Rajantie, 1981). Aversion to protein-rich food, vomiting and diarrhea, lethargy, hepatomegaly, and retarded growth and mental development are common clinical manifestations. In patients with LPI, we have observed abnormal immunological responses,probably induced by low serum arginine levels. It has been known for many years that the body produces more nitrate than is ingestedfrom the diet (Mitchell et al., 1916; Tannenbaum et al., 1978; Kurzer and Calloway, 1981). This is not entirely due to the activity of

* Corresponding author. Tel.: (+ 81-9Y) 264 2211; Fax: (+ 81-99) 265 1693. 0022-510X/95/%09.50 0 1995 Elsevier Science B.V. AI1 rights reserved SSDI 0022-510X(95)00237-5

more

nitrate

than they ingested

(Green

et al.,

1981; Witter et al., 1981; Wagner et al., 1983). It hasbeen reported that nitrate and nitrite can be synthesized by macrophages.Thus, it has been shown that nitrite production is process that occurs in mammals (Iyengar et al., 1987), both nitrate and nitrite being derived from nitric oxide (NO). Early work ruled out the likelihood of compounds such as N02-, N03-, NH,, and hydroxylamine being the sourcesof NO. Finally, the amino acid L-arginine was shown to be the precursor for the synthesis of NO by vascular endothelial cells (Palmer et al., 1988). NO participates in intercellular signaling when produced in small amounts by constitutive NO synthase (cNOS) in endothelial cells (K, [arginine], 2.9 PM; V,,,, 15.3 nmol/min/mg (Pollock et al., 1991)) and neurons (K, [arginine], 1.5 PM; V,,,, 0.96 pmol/min/mg (Bredt and Snyder, 1990)) (Moncada et al., 1991; Nathan, 1992). Immunologic and inflammatory stimuli, however, induce isoforms of NOS (inducible NOS: iNOS, K, [arginine], 32.3 PM; V,,,, 1052 nmol/min/mg (Yui et al., 1991)) that can produce much larger amounts of NO over longer periods (Nathan, 1992). Under these circumstances, NO exerts cytostatic or cytotoxic effects against microbes,

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tumor cells, macrophages, and lymphocytes, NO also suppresses the proliferation of T cells (Hoffman et al., 1990; Albina et al., 1991; Mills, 1991) and the emigration of neutrophils (Kubes et al., 1991). Thus, while diseases such as experimental acute encephalomyelitis (MacMicking et al., 1992), rabies (Koprowski et al., 1993), and graft vs. host disease (Langrehr et al., 1992) are accompanied by the induction of iNOS and the production of NO, it is difficult to predict whether the sustained generation of NO is more likely to exacerbate or to alleviate chronic inflammation. Therefore, we investigated whether the persistent low serum arginine levels and the consequently low NO levels in vivo in patients with LPI were related to their immunological status.

2. Materials

and methods

2.1. Patients In this study informed consent was obtained from each of the 3 LPI patients; all had small frames and were poorly nourished. Patient 1 was a 29-year-old woman of 150 cm and 40 kg body weight; patient 2 a 44-year-old man 165 cm tall and weighing 47 kg; and patient 3 a 47-year-old woman 148 cm tall and weighing 42 kg. Patients 1 and 2 were human T-lymphotropic virus type I (HTLV-I) seronegative, while patient 3 was an asymptomatic carrier. All patients had had syncopal episodes, similar to epilepsy, which episodes had lasted 12-24 h. Blood cell counts showed an increased number of monocytes in patient 3 and leukocytopenia in all these patients (Table 1). Liver function tests showed high levels of serum LDH, the reason not being identified (Simell et al., 1975). Serum amino acids showed high levels of serine, glutamine, glycine and alanine which acids trap ammonia, and low levels of the basic Table 1 White blood cell count, serum immunoglobulin patients with lysinuric protein intolerance

WBC/

/A(8500-4500)

Analysis of WBC granulocytes % (46-75) lymphocytes % (25-45) monocytes % (l-8) atypical cells % Immunoglobulin and complement 1gG (800-1800 mg/dl) IgA (90-450 mg/dl) IgM (60-250 mg/dl) C3 (55-120 mg/dll C4 (20-50 mg/dl)

and complement

levels in

Patient 1

Patient 2

Patient 3 a

4500

3500

4200

69 25 5 1

1410 284 160 119 27

35 59 5 1

2727 549 174 51.0 16.5

43 40 15 1

2672 247 4.59 70.0 23.4

a Patient 3 is an asymptomatic HTLV-I carrier described in detail under Results. Figures in parentheses are normal levels at Kagoshima University Hospital which were determined by a recommended procedure of IFCC (International Federation of Clinical Chemistry).

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Table 2 Serum amino acid levels in patients Values are in nmol/ml (n = 15). Patient Serine Glutamate Glutamine Glycine Alanine Citrulline Omithine Lysine Arginine

1820 b 69.7 1803 553 924 38.8 15.8 86.3 38.7

1

179

with

lysinuric

protein

intolerance.

Patient 2

Patient 3

Control

189 50.0 841 434 599 66.4 14.4 43.0 9.7

116 42.5 693 486 986 61.3 11.3 42.7 15.8

140 + 20 43.5 + 16.2 566+61 237 _+ 23 416+87 34.8 + 5.9 93.0 f 8.5 229 f 34 119+24

a

a Control subjects, 15 normal individuals of age 35 + 9 years (mean + SD) and range 23-47 years. The basic amino acids, ornithine, lysine, and arginine were decreased, while the ammonia trapping amino acids, serine, glutamine, glycine, and alanine, were increased.

amino acids arginine, ornithine, and lysine (Table 2). These findings were compatible with those of typical LPI.

2.2. Methods Serum amino acid levels were analyzed with a Hitachi Amino Acid Analyzer 835 (Hitachi Co. LTD., Tokyo, Japan) after serum had been deproteinized with sulfosalicylic acid (final concentration, 2.5%). Peripheral blood lymphocytes (PBL) were separated from heparinized peripheral blood samples by centrifugation on Mono-poly Resolving Medium (Flow Laboratories Inc., USA) carried out according to the manufacturer’s instructions. The isolated PBL were resuspended in 10% fetal bovine serum (FBS)/RPMI 1640 medium. The monoclonal antibodies (MoAb) used in this study for T cell surface markers were fluorescein isothiocyanate (FIT0 or phycoerythrin (PE)-conjugated T8 (CD8), FITC-conjugated T4 (CD41, PE-conjugated Tll (CD2), PE-conjugated I2 (HLA-DR), FITC-conjugated Mol (CDllb), PE-conjugated 2H4 (CD45RA), PE-conjugated 4B4 (CD29), FITC-conjugated CD57, and PE-conjugated CD16 (all obtained from Coulter Immunology, Hialeah, FL, USA). PBL (approximately 5 X 106) were stained with the desired MoAb at the optimal dilutions. After incubation at 4°C for 30 min, the cells were washed three times with phosphate-buffered saline (PBS) containing 0.1% NaN,, and suspended in the PBS medium, after which they were subjected to flow cytometry. The stained T cells were analyzed with an EPICS C flow cytometer (Coulter Immunology, Hialeah, FL, USA). Total lymphocytes and large lymphoblasts were identified by gating on the cytogram of the flow cytometer. The percentages of stained lymphocytes were determined on a specified fluorescence channel in comparison with the background fluorescence produced by staining with negative control mouse IgG. Leukocyte phagocytic activity was determined as follows; 100 ~1 of fluorescent beads (Fluoresbrite 2.0 pm

180

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YG, Polyscience Inc., USA) in Krebs-Ringer solution was added to heparinized blood (100 ~1) at a final ratio of beads to leukocytes of 1O:l. The mixed solution was incubated at 37°C for 60 min. The reaction was terminated by placing the reaction mixture on ice; 2 ml of lysing reagent (NH,ClS.26 g, tetrasodium EDTA 0.037 g, K,CO, 1.00 g per liter of aqueous solution) was then added to cause hemolysis. After 15 min the percentage of fluorescent to total leukocytes was determined by flow cytometry. Oxidative product formation by polymorphonuclear leukocytes (PMNL) (leukocyte cytotoxic activity) was assayed by flow cytometry under the procedure of Taga et al. (1985), modified of Bass et al. (1983). An aliquot (0.1 ml) of heparinized whole blood was added to 1.9 ml 5 PM 2’,7’-dichlorofluorescein diacetate (Eastman-Kodak Co., Rochester, NY, USA) in PBS containing 5 mM glucose and 0.1% gelatin and incubated for 15 min at 37°C. Then 500 ~1 of 25 mM EDTA and 10 ~1 of 25 pg/ml phorbol myristate acetate (Sigma Chemical Co., St. Louis, MO, USA) were added and incubated for 25 min at 37°C. After that, erythrocytes were lysed with 2 ml of cold 0.87% NH,Cl solution and the reaction solution was centrifuged for 10 min at 3000 rpm. The pellet was resuspended in 2 ml of cold PBS containing 0.1% NaN, and the ratio of fluorescent to total cells determined. Natural killer (NK) cell activity was determined by the

Table 3 Lymphocyte

subset in patients

with lysinuric

protein

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procedure of Kasahara et al. (1981) except that Ficoll-Conray solution (Daiichi Pure Chemicals, Tokyo, Japan) was employed for lymphocyte separation. Myeloid cell line, K-562 target cells (Dainippon Pharmaceutical Co. Ltd., Osaka, Japan) and effector lymphocytes were reacted for 4 h, the ratio of effector to target cells being 5O:l. Antibody-dependent cell-mediated cytotoxicity was determined as follows; one-tenth volume of silica gel LC-5H (5% solution) (Wako Pure Chemical Industries, Ltd. Osaka, Japan) was added to heparinized blood and incubated at 37°C for 1 h. Lymphocytes (effector cells) were then separated by Ficoll-Conray solution, followed by two washings with Mg2+- and Ca2+-free PBS; the lymphocytes were stored in 10% FBS/RPMI 1640 medium, at a concentration of 2.5 X 106/ml. Chicken red blood cells (CRBC) (Cosmobio, Tokyo, Japan), were prewashed twice with PBS and labelled by incubating with 100 &i of sodium [ 51Crlchromate (Daiichi Pure Chemicals, Tokyo, Japan), per 2 X lo6 CRBC at 37°C for 2 h. The CRBC were then washed twice with PBS, and suspended with 10% FBS/RPMI 1640 medium at a concentration of 5 X lo6 cells/ml. Ten ~1 of labelled CRBC and 2 ~1 of rabbit anti-CRBC antibody (Organon Teknika, NV, USA) were placed into the well of a microplate Falcon 3072 (Becton Dickinson, Oxnard, CA, USA) after which 200 ,ul of effector lymphocyte suspension was added. Incubation

intolerance

Marker

Patient 1

Patient 2

Control bt=7)

CD4 (o/o) CD8 (%I CD2 (o/o) CD4DR (%) CD8DR (or,) CD44B4 (%) CD42H4 (o/o) CD8CDllb + (%) CD8CDl lb - (%) CD57 + CD16 + (%) CD57 + CD16 - (%) CD57-CD16 + (%) T/B (ratio) CD4/CD8 (ratio) CD4DR/CD4(%) CD8DR/CD8 (%) CD44B4/CD4 (%) CD42H4/CD4 (%) CD42B4/CD42H4(ratio) CDllb+/CDllb(%)

27.4 29.8 56.8 1.9 2.2 18.2 9.5 12.1 23.6 0.3 19.0 2.9 3.4 0.9 7.1 7.4 66.5 34.8 1.9 51.2

25.4 35.6 77.1 3.5 3.2 17.2 8.0 9.6 26.9 2.0 13.0 8.0 6.2 0.7 14.0 9.0 67.7 31.5 2.2 35.7

33.7 f 6.7 30.7+5.9 75.0 f 4.4 3.1*0.9 2.6+1.8 17.3 + 5.3 15.1+ 4.0 11.3+4.3 20.2 f 6.9 8.4 + 6.3 f 10.8 f 6.9 f 3.8 f 2.4 f 4.8 f 1.2 1.2+0.4 9.3 f 3.3 8.lk5.1 50.9 + 9.0 45.0 f 6.9 1.1 f0.2 74.2 k 61.3

d

g j ’

178-182

’

h k ’

1 a

Patient 3 b 30.7 52.8 84.6 9.3 10.8 25.9 14.7 20.2 31.8 1.0 20.0 4.0 9.3 0.6 30.2 20.4 84.3 47.9 1.8 63.5

* *

*

* * i ’

Control (n=7)

2 ’

4O.lk5.4 22.4*5.1 73.7 f 7.7 4.8+ 1.9 2.4+ 1.1 18.2k7.4 18.3 + 7.2 11.3*3.1 11.9k4.8

4.9+ 1.9 1.9 f 0.5 12.1 f5.5 10.7 f 4.4 46.4 f 21.3 44.7 _+ 14.7 1.0+0.4 109.7 f 50.2

a Non-HTLV-I carrier normal control subjects (mean f SD), aged 28-48 years (36 + 8 yrs); b patient 3 (HTLV-I asymptomatic carrier) and ’ HTLV-I asymptomatic carrier control subjects (mean+ SD), aged 33-64 years (54 f 11 yrs); f normal control subjects whose HTLV-I serum titer was not determined (mean f SD, n = 50) aged 18-66 years (38 + 15 yrs). * The rate of CD4DR+ cells (activated cells), CD8DR+ cells (activated cells) and CD8CDllb - cells (cytotoxic T cells) was increased probably because patient 3 was a HTLV-I carrier, although control subjects were also carriers. The rate of lymphocyte subsets of CD42H4 (suppressor inducer) was slightly decreased (d - 1.4 SD; e - 1.8 SD). The ratio of CD44B4 + (helper inducer) to CD4 + cells was sliqhthtly increased (s 1.7 SD; h 1.9 SD; i 1.8 SD) and that of CD42H4 + to CD4 cells was slightly decreased (j - 1.5 SD; k - 2 SD), relative to controls. The ratio of CD44B4+ to CD42H4 cells was prominently increased by more than 2 SD. These findings indicate that humoral immunity was increased.

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was carried out for 20 h at 37°C and the incubation supematant was then assayed with a gamma-counter (Minaxi autogamma 5000, Packard Instruments Co. Inc., Grove, IL, USA) and the ratio of released “Cr to total 51Cr was determined. Lymphocyte proliferative responses to mitogens was determined as follows; PBL, 1 X lo5 in 200 ~1 of 10% FBS/RPM 1640 medium, were cultured in triplicate with or without the mitogens, concanavalin A (Con A; final concentration 5 pg/ml; Boehringer, Mannheim), pokeweed mitogen (PWM; final concentration 10 pg/ml; Gibco) or phytohemagglutinin (PHA; final concentration 10 pg/ml; Difco) in a microtiter plate at 37°C. After 72 h, 0.25 &i of [methyl-3H]thymidine (Daiichi Pure Chemicals) was added to each well, and the cells were harvested 16 hours after this addition. L3H]Thymidine incorporation was determined with a liquid scintillation counter (Ichikawa et al., 1982). Other chemicals used were obtained from Wako Pure Chemicals. Normal control subjects were employed in each examination, and they are described in each table legend. The examinations of leukocyte phagocytic, cytotoxic, NK cell, ADCC activity and lymphocyte proliferation were not related to the ages of normal control subjects. In regard to lymphocyte subset (Table 31, as control subjects both HTLV-I seronegative normal individuals and HTLV-I seropositive asymptomatic carriers were employed because HTLV-I causes persistent infection of CD4 positive lymphocytes, resulting in lymphocyte activation.

3. Results Serum IgG was increased in patients 2 and 3, compared to non-HTLV-I carrier or HTLV-I asymptomatic carrier (Table l), although patient 3 was a carrier. Generally in carriers serum IgG levels are high, as are levels in patients with HTLV-I associated myelopathy/tropical spastic para-

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181

paresis (HAM/TSP) (1870 k 524 mg/dl (mean k SD), n = 50; normal range, 800-1800 mg/dl at our hospital) (Osame et al., 1987). HAM/TSP is a chronic progressive HTLV-I infectious myelopathy clinically presenting a paraplegia with neurogenic bladder and mild sensory disturbances characterized by mononuclear cell infiltration in spinal cord (Osame et al., 1986). As HAM/TSP is an intensive HTLV-I infection and the detection of integrated HTLV-I proviral DNA with lymphocyte DNA indicates that the amount of HTLV-I provirus is greater in patients with HAM/TSP than in asymptomatic carriers (Yoshida et al., 19891, serum IgG levels in asymptomatic carriers are high, but not as high as those in patients with HAM/TSP. Even compared to the IgG level of patients with HAM/TSP, the serum IgG level in patient 3 was high. In regard to lymphocyte subpopulations (Table 31, the ratio of CD4DR positive cells, that of CD8DR positive cells (activated cells) and that of CD8CDllbcells (cytotoxic T cells) were increased in patient 3 because patient 3 was a HTLV-I carrier; however, compared with HTLV-I seropositive control subjects, these ratios were increased. The reason may be due to more serious HTLV-I infection in patient 3 than the control subjects, otherwise due to low serum arginine level. The ratio of CD42H4 positive cells (suppressor inducer) to CD4 positive cells in patients 1 and 2 was slightly decreased compared to controls and the ratio of CD44B4-positive cells (helper inducer) to CD4 positive cells was slightly increased. The ratio of CD44B4 positive cells to CD42H4 positive cells was increased. The ratio of CD57-CD16 + cells which have strong NK cell activity, and that of CD57 + CD16 - cells, which have weak NK activity, was not changed. The ratio of CD57 + CD16 + cells, which have moderate NK cell activity, was slightly decreased, compared to controls. NK cell activity was markedly decreased and leukocyte phagocytic and cytotoxic activities were also decreased, compared with controls, while ADCC activity was within the normal limits (Table 4). The results had not been

Table 4 Immunological examination of patients with lysinuric protein intolerance. Normal control levels indicate mean+SD. Normal control levels were a SO.l+ 13.4% (mean+SD, n = 66) of women, ages 22-59 years (37+ 11 yrs); b 59.8k 15.7% (n = 69) of men, ages 22-58 years (37+11 yrs). Leukocyte cytotoxic activity, and NK cell activity were decreased, these were related to the low serum arginine level, while ADCC (antibody-dependent cell-mediated cytotoxicity) activity was not decreased. Lymphocyte proliferation in the absence of PWM, Con A, or PHA was increased in patients 1 and 2. Patient 3 was an asymptomatic HTLV-I carrier and is described under Results. Activity

Control

Leukocyte phagocytic activity (%) Leukocyte cytotoxic activity (%) NK cell activity (%) ADCC activity (%I Lymphocyte proliferation * * * PWM(cpm) PWM+ (cpm) Con A - PHA - (cpm) Con A + (cpm) PHA+ (cpm)

82+5 95&i

*

n = 16, ages 24-51

Patient * *

56.7+7.8

* *

894+534 27099k13226 420+237 48361k13886 60435+19435

years (37 + 11 yrs, mean + SD). * *

n = 66, ages 19-48

Patient 2

Patient 3

50 85 2.5 a 76

1

28

68 82 5.8 a 60

3542 16208 16948 63214 59000

4408 26076

81 39.8 75

b

1494 43587 43587

years (33 f 10 yrs). * * *

n = 52, ages 21-59

4764 36449 2264 60672 50921

years (37+

11 yrs).

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recognized in the HTLV-I asymptomatic carrier; however, NK cell activity in patients with HAM/TSP was shown to be decreased (Osame et al., 1987). NK cell activity in patients with HAM was found to be 15.8 f 10.7% (n = 111, while the value was 29 + 11% (n = 50) in normal controls. These findings were derived from an assay procedure that differed from ours, and were markedly lower than ours; however, our findings were suggestive of decreased NK cell activity in patient 3. Lymphocyte proliferation induced by PWM, Con A or PHA was not changed (Table 4); however, lymphocyte proliferation in the absence of PWM, Con A, or PHA was increased. Although this phenomenon indicates the spontaneous proliferation of lymphocytes in an asymptomatic HTLV-I carrier, such as patient 3 (Itoyama et al., 1988), such proliferation was also recognized in patients 1 and 2.

result of the decreased cellular immunological activity brought about by the decreased NO production, humoral immunological activity, conversely, appears to increase to maintain immunological protection. Although the high level of serum ammonia appears to be responsible for manifestation of the signs and symptoms in patients with LPI, it also appears that the persistently low level of NO in vivo, derived from the persistently low level of arginine, is related to the manifestation of the signs and symptoms. Our third patient had a syncopal episodes associated with slight left hemiplegia with positive Babinski sign in the absence of particularly high levels of serum ammonia, and disappeared soon after admission to our hospital. Since NO is a very strong vasodilator, the relation of the low NO level to the manifestation of the signs and symptoms should be explored.

4. Discussion

References

Nitric oxide (NO) is a mediator in the cytotoxic activity of macrophages, leukocytes and NK cells against tumor cells and invading bacteria and protozoa. NO produced from arginine and the successively formed free radicals is implicated in this cytotoxic activity (Liew and Cox, 1991: Chan et al., 1992; Charles et al., 1992; Beckerman et al., 1993). Patients with LPI associated with low serum arginine and lysine levels probably produce low levels of NO, resulting in low cytotoxic activities of NK cells and leukocytes. However, the activity of antibody-dependent cellmediated cytotoxicity (ADCC) was not low in this experiment, the reason for this being that we determined ADCC activity by monitoring the hemolysis of chicken red blood cells. In this process, NO may not be as closely associated with hemolysis as perforin, a protein with strong hemolytic activity that is secreted by effector cells (Young and Cohn, 1988). NO is involved with immunological regulation, and it has been demonstrated that suppressor macrophages and lymphocytes produce NO, and inhibit lymphocyte proliferation (Ichikawa et al., 1982; Albina et al., 1991; Mills, 1991; Kawabe et al., 1992; Schleifer and Manfield, 1993). These findings are compatible with our findings that lymphocyte proliferation in the absence of mitogen was increased in patients 1 and 2, although in this experiment lymphocyte proliferation induced by mitogens (PWM, Con A, and PHA) was not increased. The ratio of suppressor inducer lymphocytes (CD42H4 positive) to CD4 cells was slightly decreased and, conversely, the ratio of helper inducer lymphocytes (CD44B4 positive) to CD4 cells was slightly increased. These results brought prominently increased ratio of CD44B4 to CD42H4. This altered balance was probably responsible for the increased serum IgG level. In addition, this findings may be simply accounted for by the effect of the low NO level of suppressor macrophage and lymphocytes. As a

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