Acta Med Scand 1985; 218: 251-5

Immunological Studies in a Patient with the Glucagonoma Syndrome

MIKAEL DOHLSTEN and TORGNY HALLBERG From the Department of Medical Microbiology, Lund University, Lund, and the Clinic of Geriatric Medicine, County Hospital, Halmsrad, Sweden

ABSTRACT. Dohlsten M, Hallberg T. (Department of Medical Microbiology, Lund University, Lund, and Clinic of Geriatric Medicine, County Hospital, Halmstad, Swe- den.) lmmunological studies in a patient with the glucagonoma syndrome. Acta Med Scand 1985; 218: 251-5.

The glucagonoma syndrome is a rare clinical entity characterized by a glucagon-producing tumor of the pancreas, necrolytic migratory erythema, weight loss and usually decreased glucose tolerance. Lately there has been increasing interest in the interaction of peptide hormones and the immune system, implicating a regulatory role of the peptide hormones on immune activation and function. We present a patient with metastasizing glucagonoma and highly elevated plasma levels of glucagon and pancreatic polypeptide. Normal num- bers of peripheral blood lymphocytes with normal proportions of the T and B populations were seen. Within the T lymphocyte population the percentage of T4+ cells (helper phenotype) was increased with a concomitant decrease in T8+ cells (suppressorkytotoxic phenotype), resulting in an abnormally high T4R8 ratio of 6.7 (mean reference value 1.8). Functional tests demonstrated a normal tuberculin reaction and adequate lymphocyte responses in vitro to polyclonal activators. Furthermore we noticed a urine electrophoretic pattern consistent with a proximal tubular kidney defect. It is concluded that studying the immune system in patients with endocrine active pancreatic tumors may give clues on the influence of pancreatic peptide hormones on immune function and regulation. Key words: glucagonoma, glucagon, pancreatic polypeptide, T lymphocytes, B lymphocytes, urine electrophoresis.

Hypersecretion of glucagon causes a distinctive clinical syndrome with a peculiar skin rash termed ‘necrolytic migratory erythema’, glucose intolerance or manifest diabetes, weight loss, and hypoaminoacidemia. In addition, hypersedimentation, hypoproteinemia and glossitis may occur ( I , 2). Most glucagonomas are malignant and metastasize to the liver but the tumors are often slow growing (1, 2). The primary tumor is usually located to the tail of the pancreas corresponding to the frequency of a-cells in normal pancreas (2). Glucagonomas may secrete multiple hormones like insulin, adrenocorticotropic hormone (ACTH), pancreatic polypeptide (PP), serotonin or ‘vasoactive intestinal peptide’ (VIP) ( 2 , 3).

In the last years increasing interest has been focused on the effect of polypeptide hormones on the immune system. ACTH has been shown to play a regulatory role in antibody production by B lymphocytes as well as for interferon production by T lympho- cytes in vitro (4). Human peripheral blood lymphocytes express specific binding sites for VIP and accumulate cyclic adenosine monophosphate upon stimulation with VIP ( 5 ) . Activated T lymphocytes have been reported to express receptors for insulin and these receptors have been suggested to be of importance in activation of the T cells (6). Also,

Abbreviations: ACTH=adrenocorticotropic hormone, PP=pancreatic polypeptide, VIP=vasoactive intestinal peptide, PHA=phytohemagglutinin, PWM=pokeweed mitogen, Th=T helper cells, Tsc=T suppressor/cytotoxic cells, Tm and Tg=T cells with receptors for the Fc part of IgM and IgG, respectively.

252 M. Dohlsten and T. Hallherg Acta Med Scand 1985; 218

Fig. I Fig. 2

b I I

Fig. 1 . Computed tomography of the upper abdomen showing the primary tumor, corresponding to the tail of the pancreas, and multiple hepatic metastases. Fig. 2. Agarose electrophoresis. (A) Serum electrophoresis showing a normal pattern except for moderate hypoalbuminemia. (B) Urine electrophoresis (a=albumin, b=zinc a-2-glycoprotein 1, c=re- tinol-binding protein, d= transfemn, e=B-2-microglobulin).

glucagon receptors have been detected on lymphocytes activated by either T cell growth factor (interleukin-2) or phytohemagglutinin (7). These receptors were expressed simulta- neously with insulin receptors but the two receptors occurred in different concentrations, depending on the stimuli used for lymphocyte activation.

A possible role for the glucagon receptor on lymphocytes has not been proposed so far. We have analysed the distribution of differentiation antigens on peripheral T and B lymphocytes as well as the ability of the lymphocytes to respond to mitogen stimulation in a patient with glucagonoma. The patient had highly elevated plasma glucagon for at least one year prior to the immunological study.

CASE REPORT A 47-year-old man was admitted to hospital due to heavy weight loss (down to 36 kg) in the last two years. The patient also had a nonspecific dermatitis on the legs as well as a facial erythema with papules. He had no family history of malignant diseases, dermatological disorders or diabetes.

Computed tomography of the abdomen showed multiple hepatic metastases and a tumor corre- sponding to the tail of the pancreas (Fig. I ) . He had no diarrhea or steatorrhea. During hospitalization the patient developed extended skin lesions with erythematous erosions, eruptions, crusts and scaling. The lesions were migrating and most healed areas were hyperpigmented. The clinical feature of the lesions and histological examination of a skin biopsy specimen gave a picture consistent with necrolytic migratory erythema as seen in glucagonoma. The skin lesions responded well to steroid therapy and the patient was placed on a low-dose continuous steroid therapy (prednisone 10 mg/day). After 4 months in the hospital the patient was treated with dacarbazine, 220 mglday for 5 days. The treatment was then discontinued because the patient refused further chemotherapy.

The patient developed hyperglycemia after 5 months’ stay in the hospital. The hyperglycemia soon became insulin-dependent and was accompanied by glucosuria. No further active treatment was given, the tumor mass remained unchanged during the following year and the clinical condition

Acta Med Scand 1985; 218

actually improved. The immunological examinations were carried out after 16 months’ hospitaliza- tion, during which time the patient had continuously elevated plasma glucagon values. The patient w a s discharged soon thereafter on his own request.

Plasma glucagon values measured by a radioimmunoassay were 430-280 pmol/l during the 18 months of hospitalization (reference value <60). The serum PP level was highly elevated, 2 700 pmoUl (reference value <30). Insulin, somatostatin, ACTH, and VIP analyses were normal. Serum levels of creatinine, sodium and potassium were normal. Erythrocyte sedimentation rate was elevated, normo- cytic normochromic anemia was present and plasma amino acids were decreased. Except for moderate hypoalbuminemia, serum electrophoresis was normal including normal levels of IgG, IgM and IgA.

Agarose urine electrophoresis (performed at the Department of Clinical Chemistry, Malmo General Hospital, Malmo) showed slight protein excretion, 0.18 g/l (reference <O.l), with a urine albumin content of 0.05 g/l (reference <0.025), and increased concentrations of several low molecular weight proteins (Fig. 2). Zinc a-2-glycoprotein and retinol-binding protein were detected in the a-2 zone, and quantitation of urine /?-2-microglobulin by radioimmunoassay revealed highly elevated values, 28 mg/l (reference <0.6). The result of the analysis of urine proteins was consistent with a proximal tubular defect of the kidney.

The patient had normal white blood cell counts (6.77x109/1) and the differential count was also normal (N 64, E3, B 1, L26, M5). The patient had been BCG-vaccinated 30 years ago, his PPD test w a s now strongly positive at 2 TU, 13x14 mm (>6 mm is regarded as a positive reaction).

Peripheral blood lymphocytes were classified by indirect immunofluorescence using monoclonal antibodies OKT 3 (pan T cells), OKT 4 (mainly T helper cells (Th)), OKT 8 (mainly T suppressorky- totoxic cells (Tsc)), OKT 11 (T cell marker: sheep red blood cell receptor), all from Ortho Diagnostic Systems, and monoclonal antibody HB-31 (B cell antigen) from Miles-Yeda. Furthermore, the presence of membrane immunoglobulin (not class-restricted) and membrane IgM on B lymphocytes were analysed using fluorescein-conjugated F(ab’)2-fragments of goat-antihuman Ig reagents (Kalle- stad). All fluorescent antibody tests were read in the microscope. The expression of the sheep red blood cell receptor on T lymphocytes was also demonstrated by conventional rosetting technique (Table I). Methodological details have been published elsewhere (8).

The capacity of lymphocytes to respond to polyclonal mitogen stimulation was studied using phytohemagglutinin (PHA) (HA15, Wellcome) and pokeweed mitogen (PWM) (Gibco Laboratories) in a 3-day in vitro culture (Table 11). The cultivation procedure of Stenstam et al. (8) was slightly modified.

Immunological studies in glucagonoma syndrome 253

RESULTS AND DISCUSSION

Our patient had a history of weight loss, necrolytic migratory erythema, diabetes, hypo- aminoacidemia, hypoalbuminemia, normocytic normochromic anemia, elevated sedimen-

Table I. Cell surface markers on peripheral blood lymphocytes (% of reactive cells)

Reference value“ Patient’s value Mean Range

B lymphocytes Membrane Ig (total) Membrane IgM HB-31 antigen

OKT 3 (pan T) OKT 11 (pan T, SRBC receptor) S R B C ~ rosetting OKT 4 (T helper) OKT 8 (T suppressorkytotoxic) T4+/T8+ cell ratio

T lymphocytes

5.0 1.5 2.5

81 81 65 65 9.5 6.7

11.2 5.8 5.4

18 86 69 50 30

1.8

3.5-19 3.5-10 1.9-13

59-89 75-92 55-83 38-58 17-38 1.0-3.4

a From 10-12 healthy adults. Sheep red blood cells. n-31.

254 M . Dohlsten and T . Hallberg Acta Med Scand 1985; 218

tation rate and highly elevated glucagon and PP levels, a picture consistent with the glucagonoma syndrome (2). Although the patient recieved no continuous active treatment, his clinical picture improved and his tumor mass remained constant as judged by repeated computed tomographies.

Results obtained by monitoring the general immunological capacity of patients with malignant diseases are often difficult to evaluate on a prognostic or cause-effect basis. We conclude that several tests dependent on functioning Th cells (cutaneous tuberculin hypersensitivity, PHA activation and PWM activation (T cell dependent B cell mitogen)) worked normally in this patient (Table 11). Also, the B cell limb of the immune system presented an overall functioning picture with normal levels of IgG, IgM and IgA and normal or slightly decreased levels of peripheral blood B lymphocytes marked by mem- brane immunoglobulins or B cell antigen HB-31 (Table I).

The salient observation in the immunological study was the elevated T4/T8 balance with more than 3-fold increased ratio compared with normal individuals (Table I). Depression of the Th/Tsc balance has been demonstrated in a number of diseases. In some of these, most notable in the acquired immunodeficiency syndrome, the alteration is directly linked to the pathogenesis of the disease. Low Th/Tsc ratios may, however, be transient-one example being cytomegalovirus infections (9). Increased T4/T8 ratio has been seen in some autoimmune diseases including pernicious anemia (10).

The functional importance of the T4/T8 imbalance for the course of the disease in our patient can only be speculated upon. However, more open to further analysis is the question whether or not it is linked to long-standing abnormal plasma levels of glucagon and/or PP. We were not able to demonstrate any effect of the patient's serum on the maximal mitogen response of either patient or normal lymphocytes (Table 11).

Suppressor T cells may interfere with successful tumor defences in certain animal systems ( I 1). Attempts to translate such observations to the human T4 and TS populations should, however, be made with great caution. It must be emphasized that the T4 and T8 populations are functionally heterogenous and not identical with T helper and T suppres- sor cells, respectively. Thus, certain T suppressor cells are contained within the T4+ phenotype (12) and, equally important, cytotoxic T lymphocytes are included in the T8+ phenotype.

Advanced protein calorie deficiency is accompanied by derangements of the individual's immunological capacity, where the T part seems to be more sensitive than the B part (13). Low T cell numbers, low PHA response and a lowered Tm/Tg lymphocyte ratio have thus

Table 11. Mitogen stimulation of patient and control lymphocytes (cpm 3-H-thymidine uptake x Tissue culture medium (TCM) was supplemented with 15 % of either fetal bovine serum (FBS), human AB serum (AB), serum from the patient (P), or serum from a healthy control (HC). The test was performed with a series of PHA and PWM dilutions. 3-H-thymidine uptake in the absence of mitogen (0 .2 -1 .5~ lo3 cpm) not subtracted

Maximal PHA response Maximal PWM response TCM containing P cells HC cells P cells HC cells

FBS 175 210 32.2 11.4 AB 192 241 58.1 23.1 P I68 213 23.8 15.5 HC 160 222 38.8 17.8

Acta Med Scand 1985; 218

been observed in nutritional deficiency (13). These alterations are not consistent with our patient’s clinical picture. Moreover, the gross initial malnutrition was mainly reversed at the time of the immunological study.

Administration of prednisone to healthy subjects may cause peripheral lymphopenia largely accounted for by a reduction in circulating OKT I 1 + and OKT 4+ cells (14). This pattern is rather the opposite to that seen in our patient and contradicts the importance of prednisone therapy for the alteration in lymphocyte subsets.

Furthermore we found tubular proteinuria in our patient. He had glucosuria, normal serum creatinine, and urine electrophoresis showed excretion of several low molecular weight proteins. The urine content of #l-2-microglobulin was highly elevated, a picture consistent with a defect in proximal tubuli as reported by others (15). Whether the tubular proteinuria is associated with the immunological dysfunction or represents a further feature of the glucagonoma syndrome remains an open question. We thus propose the relevance of lymphocyte subpopulation studies as well as urine electrophoresis in patients with the glucagonoma syndrome.

Immunological studies in glucagonoma syndrome 255

ACKNOWLEDGEMENTS Financial support from the Medical Faculty, Lund University, and the Swedish Medical Research Council (grant no. 5682) is gratefully acknowledged.

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Received Dec. 3, 1984.

Correspondence: T. Hallberg, Department of Medical Microbiology, Lund University, Solvegatan 23, S-223 62 Lund, Sweden.