ADONIS 0009910491002404
Clin. exp. Immunol. (1991) 85, 335-340
Immunosuppressive effects of 2-acetyl-4-tetrahydroxybutyl imidazole (THI) in the rat S. J. P. GOBIN & J. A. PHILLIPS DH Department of Toxicology, St Bartholomew's Hospital Medical College, London, England
(Acceptedfor publication 21 February 1991)
SUMMARY THI is a component of ammonia caramel, a widely used food colouring. The effect of THI on the immune system has been determined in the male F344 rat. THI was given in the drinking water at doses of 1, 10 and 50 mg/l (equivalent to 0-1, 1 and 5 mg/kg per day) to animals on a vitamin B6deficient diet. After I week, the immune competence of the animals was assessed under continued THI treatment. No marked changes in thymus or spleen weight were observed after THI treatment, although there was an increased number of pyknotic cells in the thymic cortex, mainly engulfed by macrophages and there appeared to be a slight thinning of the cortex area. THI produced a significant loss in T and B lymphocytes in peripheral blood but not in the spleen. No change in natural killer (NK) cell activity against YAC-1 target cells was observed in the spleen. The observed increase in NK cell activity in peripheral blood was due to an increase in circulating large granular lymphocytes (LGL). Although the serum antibody titre against keyhole limpet haemocyanin (KLH) was not affected by THI treatment, B cells showed less proliferation when cultured with lipopolysaccharide. T cell function was impeded as measured in mitogen-induced proliferation assay, delayed-type hypersensitivity assay and host versus graft (popliteal lymph node) assay. The results indicate that THI is an immunosuppressor in the rat, in whom it can produce profound lymphopenia and suppression of cell-mediated immunity.
Keywords 2-acetyl-4-tetrahydroxybutylimidazole immunosuppressors lymphopenia cell-mediated immunity natural killer cells INTRODUCTION Caramels at one time constituted 95% by weight of all the colouring materials used in human food stuff (Patey et al., 1985). One type of caramel (ammonia caramel) is prepared by controlled heating of carbohydrates in the presence of ammonia. In oral toxicity studies, ammonia caramel has been shown to reduce the number of peripheral blood lymphocytes in rats, without evidence of other toxic effects (Evans et al., 1977; Gaunt et al., 1977). Recently, the active component has been identified as THI (Kroplien et al., 1985). THI produces a rapid lymphopenia in rats, which arises within 16 h after a single dose and is completely reversible within 48 h (Phillips & Paine, 1990). There is a gradual reduction of the number of circulating TH, T,/, and B lymphocytes after treatment at doses as low as 1 mg/kg per day (Gobin et al., 1989). In addition, it was found that the effect of THI is inhibited by B6 vitamers (pyridoxine, pyridoxal and J.A.P. present address: Research Centre, Ciba-Geigy Pharmaceuticals, Horsham, UK. Correspondence: S. J. P. Gobin, DH Department of Toxicology, St Bartholomew's Hospital Medical College, Dominion House, 59 Bartholomew Close, London EC1 7ED, UK. 335
pyridoxamine) administered in the diet or parenterally (Gobin & Paine, 1989). The biological significance of the lymphopenia is unclear, but redistribution of circulating lymphocytes to the bone marrow (Fauci, 1975) and the resulting lymphopenia are important factors in the immunosuppressive effects of glucocorticoids (Bloemena et al., 1990). The present study was an investigation into the effects of THI on immunocompetence in the rat. MATERIALS AND METHODS Animals Male Fischer 344 rats, body weight 175-200 g, were purchased from Charles River, Margate, UK. They were maintained in airconditioned, light-regulated rooms (12 h light/dark cycle), 20 + 3C and 50 + 10% relative humidity. The animals were conditioned to eating a semi-synthetic powdered diet (Rat and Mouse maintenance diet no. 1, containing 4-9 mg/kg pyridoxine; SDS, Witham, UK) from the day of arrival to 3 days before the start of the study. From day -3, the animals were given a vitamin B6-deficient diet (contain-
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ing 0 04 mg/kg pyridoxine), or, ifindicated, a normal (4 9 mg/kg pyridoxine) or a vitamin B6-rich diet (10 mg/kg pyridoxine), for the remainder of the study. The diet and drinking (tap) water were available ad libitum. Hybrid (BN x F344)Fl rats used in the host versus graft assay were bred in our own department. Male BN and female F344 rats were obtained from Charles River, Margate, UK.
Chemicals THI (batch no. MDI-55-1) was kindly provided by the International Technical Caramel Association, Washington, DC. Tissue culture materials, including special prepared pyridoxine free RPMI 1640 medium, were obtained from Flow Lab.; all other chemicals from Sigma Chemical Co., Poole, UK and BDH, Dagenham, UK.
Protocol Animals in the treatment group were given THI in the drinking water at 1, 10 or 50 mg/l and were maintained on a vitamin B6deficient diet throughout the study. Water consumption was measured and showed that the average volume drunk per rat per day was equivalent to a dose of THI of 0 1, 1 or 5 mg/kg body weight per day and subsequently are expressed as the calculated daily intake.
Histology Rats were killed by asphyxiation with CO2, and the thymus, spleen, mesenteric lymph nodes, Peyer's patches and adrenal glands were removed, weighed and fixed in 10% neutralbuffered formalin, dehydrated and embedded in paraffin wax. Sections were prepared at 5 pm thickness and stained with haematoxylin and eosin for microscopy. Haematology At necropsy blood from the inferior vena cava was collected in tubes pre-treated with EDTA as anti-coagulant (Sterilin, Hounslow, UK). The total leucocyte count was determined using a Baker 7000 Haematology Analyzer (Baker Instruments, Egham, UK). The percentage of lymphocytes was determined by counting at least 200 cells in two blood films, made from the same blood sample, stained with a modified Wright stain using a Miles Hemotek II (Miles Lab., Stoke Poges, UK). Absolute lymphocyte counts were calculated from the percentage lymphocyte and total leucocyte count. Flow cytometry Analysis of lymphocyte subsets by flow cytometry was performed as previously described (Gobin et al., 1989). Briefly, erythrocyte-lysed leucocyte suspensions from blood or spleen were stained with the primary antibody (MARKI as B cell (immunoglobulin light chain) marker, 1/500; OX19 as T cell (CD5) marker, 1/1000; Serotec, Oxford, UK) for 30 min, followed by a fluorescein-conjugated rabbit F(ab')2 anti-mouse immunoglobulin antibody (1/200; Dako, High Wycombe, UK), for 30 min. The samples were fixed in 10% neutral-buffered formalin and the labelled cells were analysed by an Ortho Cytofluorograf 2150 (Ortho Diagnostic Systems, Westwood, MA) to obtain the percentages of each lymphocyte subset. Null (N) cell percentage was defined as immunoglobulin- and CD5-
cells. Absolute subset numbers were calculated as percentage of absolute lymphocyte number.
Isolation of cells from spleen and blood The spleen was asceptically removed and pushed through a stainless steel strainer with the plunger from a 10-ml syringe. The cell suspension was left on ice for 10 min to let cell clumps and debris settle. The cells were washed in RPMI 1640 following centrifugation (300 g, 10 min) and the cell pellet was resuspended in 4 ml buffered ammonium chloride solution (0- 16 M NH4Cl, 0 01 M KHCO3, 0 1 mm EDTA, pH 7-4) to lyse the erythrocytes. The cells were washed twice more and resuspended in the appropriate culture medium used in the mitogen-induced proliferation or natural killer (NK) assay. Blood from the inferior vena cava was collected in pretreated EDTA tubes and 2 ml whole blood were mixed with 3 ml buffered ammonium chloride solution to lyse the erythrocytes, washed twice and resuspended in appropriate culture medium.
Mitogen-induced proliferation assay Spleen cell suspension from rats treated with THI for various time periods were prepared as described above and were cultured with pyridoxine-free RPMI 1640 medium containing 5% heat-inactivated fetal calf serum (FCS), 20mM L-glutamine, 50 mg/l gentamicin and 50 pM 2-mercaptoethanol (complete medium). FCS was pre-tested for cytotoxicity. In order to remove adhering macrophages, spleen suspensions were incubated for 2 h in plastic culture flasks and the supernatant was used in the proliferation assay. Spleen cells (5 x I05 in 100 pl) were added to quadruplicate wells of a flat-bottomed microtitre plate, each well containing 100 pl complete pyridoxine-free complete medium with mitogens in optimum concentrations: concanavalin A (ConA) 10 Mg/ ml, pokeweed mitogen (PWM) 10 pg/ml, and lipopolysaccharide (LPS) from Escherichia coli 0127:B8 25 pg/ml, final concentrations. The cultures were incubated for 72 h at 37°C, in 5% C02/air. The cells were pulsed with 3H-thymidine (3H-TdR, 2 0 Ci/mmol, Amersham International, Amersham, UK), I pCi/ well, for 4 h before being harvested on glass fibre filter with a cell harvester (Ilacon, Tonbridge, UK). Incorporated radioactivity was counted in a scintillation counter (Canberra Packard, Pangbourne, UK) and expressed as ct/min. In other experiments, THI was added to the medium at a concentration of 0 1, 0 5, 1-0, 5, 25 and 100 pg/ml. NK assay NK activity was measured as cytotoxicity against YAC- I cells, a murine T cell lymphoma. Target cells (5 x 106) were labelled with 50 pCi Na25"CrO4 (10-35 mCi/mmol; Amersham International) for 1 h at 37°C, followed by three washes in warm RPMI 1640 and were resuspended at 105/ml complete medium (standard RPMI 1640, containing 1 mg/I pyridoxine, 10% FCS, 20 mM L-glutamine, 50 mg/l gentamicin). Target cells were added at 104 cells in 100 pl to effector cells (measured as total leucocyte number) in quadruplicate wells in round-bottomed microtitre plates, to give various effector-to-target (E:T) cell ratios. The plates were centrifuged at 300 g for 1 min and incubated for 4 h at 37°C, in 5% C02/air. The plates were then centrifuged at 300 g for 5 min and 100 p1 supernatant of each well were counted for isotope release in a gamma counter
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Immunosuppressive effects of THI (Minaxi 5530, Packard, Pangbourne, UK). The percentage specific lysis was calculated as:
Table 1. Relationship between dietary pyridoxine (vitamin B6) content and degree of lymphopenia after 7 days of TH I treatment
Experimental - Spontaneous release 100% Maximum -Spontaneous release Maximum release was determined using the supernatant from target cells treated with I M HCI 100 p1. The control wells for determining spontaneous release contained target cells with medium only.
Antibody production Antibody titre was measured after challenge with keyhole limpet haemocyanin (KLH), as described by Exon, Talcott & Koller (1986). Animals were injected subcutaneously with 1 mg KLH in 0-2 ml saline on days 8 and 15 after the start of THI treatment. On day 22, blood serum was collected and IgG and IgM titres to KLH antigen were determined by ELISA. A 96-well microtitre plate was coated overnight with 2 mg/ml KLH. Serum samples were diluted and added to wells of the KLH-coated microtitre plate. The serum samples were incubated for I h, removed, the plates were washed, and then dilutions of an alkaline phosphatase-conjugated goat anti-rat IgG or goat anti-rat IgM antibody were added and incubated for 1 h at room temperature. After adding the substrate (p-nitrophenylphosphate, 1 mg/ml in diethanolamine buffer) the colour reaction was measured after 15 min at 405 nm on a Titretek Multiskan MC (Flow Lab., Rickmansworth, UK). Results are expressed as log antibody titre. Delayed-type hypersensitivity (DTH) assay The DTH reaction was measured after 7 days of THI treatment (1 mg/kg per day) as ear swelling by a slight modification of the method of Henningson et al. (1984). Rats were sensitized with 100 pg bovine serum albumin (BSA) resuspended in 100 p1 Freund's complete adjuvant (FCA) administered subcutaneously at one side in the intrascapsular space and THI treatment continued. Rats serving as negative control were injected with 100 p1 saline and failed to show a DTH reaction after challenge. Seven days later the rats were challenged with 2% heat-aggregated BSA 30 pl injected into the pinna of the left ear and 30 p1 saline as a control in the right ear. DTH reaction was measured 24 h after challenge by measuring the thickness of each ear using a micrometer with 10-pm gradations. The average of two measurements was taken. Ear swelling is expressed as the difference of the thickness increase between the challenged ear and the control ear over 24 h. Host
versus
graft (popliteal lymph node)
assay
A host versus graft reaction was induced in the popliteal lymph node by injecting allogeneic (BN x F344)F1 spleen cells into a parental strain rat (Ford, Burr & Simonsen, 1970; Rolstad, 1985). Into each hind foot-pad of an F344 recipient rat, 107 cells in 0-1 ml PBS were injected, 8 days after the start of THI treatment. Three days after the challenge both popliteal lymph nodes were removed, weighed, pooled and a cell suspension was prepared by pushing the nodes through a stainless steel strainer. Cell numbers per lymph node were determined by counting them in a Baker 7000 Haematology Analyzer (Baker Instru-
ments).
Lymphocytes (106/ml) (Pyridoxine content (mg/kg)) THI (mg/kg per day)
0 0-1 10 5-0
0-04
4-9
10 0
7-8+0 9 4-6+1-4* 2-4+0-6* 2.5+1.1*
6-6+0-2 5-1+0.5* 3-2+0 5* 3.4+0.9*
6-9+0 7 8-1+1-1 6 5+ 1 5 4.5+1.0*
Mean+s.d. (n=6). * Significance of difference from control (P < 0-001). Table 2. Effects of 10 days of THI treatment and adrenal weight Dose (mg/kg per day) 0 0.1 1.0 50
on
body, thymus, spleen
Body weight (g)
Thymus (mg)
Spleen (mg)
Adrenal (mg)
260+11 272+8 261 +14 258+ 15
394+57 403+90 359 + 80 332+81
560+19 576+14 654 + 50* 762+39*
49+11 47+14 47 + 14 45+2
Mean + s.d. (n = 6). * Significantly different from control (P < 0 001).
Statistical analysis The significance of differences between means was determined by Student's t-test. Differences between experimental and control groups were considered to be statistically significant at P
