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Histamine contamination of pokeweed mitogen
Journal of Immunological Methods, 55 (1982) 355-360
355
Elsevier Biomedical Press
Histamine Contamination of Pokeweed Mitogen James M. Seltzer, Richard D. O'Connor 1, Ronald A. Simon and Tony E. Hugli Immunology and Allergy Division, Department of Pediatrics, School of Medicine, University of California, San Diego, La Jolla, CA 92093, and Division of Allergy and Clinical Immunology, and Department of Molecular Immunology, Scrzpps Clinic and Research Foundation, La Jolla, CA 92037, U.S.A.
(Received 26 May 1982, accepted 18 June 1982)
The histamine content of commercial preparations of pokeweed mitogen was measured by amino acid analysis technique, automated fluorometry, and bioassay employing the guinea pig ileum. Ten samples from 5 companies were examined and found to contain between 0.026 #g and 167.5 #g of histamine per ml of solution. The protein content of 9 of these putative 5 mg samples measured by folin assay and by amino acid analysis varied from 0.56 to 4.4 mg. Their amino acid compositions were similar, except for notable variations in 3 of the 16 residues quantitated. Key words: pokeweed mitogen - - histamine - - lgE
Introduction P o k e w e e d m i t o g e n ( P W M ) is an extract of the weed P h y t o l a c c a A m e r i c a n a that stimulates b o t h B a n d T l y m p h o c y t e s in vitro (Douglas, 1972; J a n o s s y a n d Greaves, 1972). This s u b s t a n c e has b e e n used p r i m a r i l y to s t u d y in vitro m o n o n u c l e a r cell p r o l i f e r a t i o n a n d i m m u n o g l o b u l i n (Ig) synthesis. Samples available from c o m m e r c i a l sources are p u r i f i e d b y a variety of different techniques. R e c e n t studies have shown that h i s t a m i n e inhibits m o n o n u c l e a r cell p r o l i f e r a t i o n i n d u c e d b y the p l a n t m i t o g e n s c o n c a n a v a l i n A ( C o n A) a n d p h y t o h e m a g g l u t i n i n ( P H A ) . This effect a p p e a r s to be m e d i a t e d b y l y m p h o c y t e s b e a r i n g h i s t a m i n e type 2 r e c e p t o r s on their surface ( W a n g and Z w e i m a n , 1978; R o c k l i n a n d H a b e r e k - D a v i d son, 1981). M o n o n u c l e a r cells f r o m a t o p i c p a t i e n t s with extreme elevations of IgE have d e m o n s t r a t e d p o o r proliferative responses to P W M ( O ' C o n n o r et al., 1980). Since these results were o b s e r v e d using a whole b l o o d culture technique that preserves b a s o p h i l function, we speculated that P W M might serve as an allergen causing I g E - m e d i a t e d release of histamine. T o test this p o s s i b i l i t y the h i s t a m i n e
1Address reprint requests to Richard D. O'Connor at U.C.S.D., School of Medicine, Immunology and Allergy Division, Department of Pediatrics (M-009-D), La Jolla, CA 92093, U.S.A. 0022-1759/82/0000-0000/$02.75
© Elsevier Biomedical Press
356
concentration of PWM-stimulated cell culture supernatants was measured. Large amounts of histamine, determined by radioisotopic enzymatic assay, were found in supernatant fluids from cell cultures as well as in cell-free control samples which contained only PWM. We then examined the histamine content of PWM samples available from several commercial sources to determine if PWM could be the source of histamine.
Materials and Methods
Preparation and storage of P WM Ten 5 mg samples of PWM obtained from 5 companies were analyzed for the presence of histamine. Each sample represents a different lot number. All samples were diluted in 1 ml sterile phosphate-buffered saline, and stored at - 2 0 ° C until assayed. Determination of h&tamine content Three methods were used to document reliability of our results: automated fluorometry modified from Siraganian (1974); histamine-induced contraction of guinea pig ileum smooth muscle (Cochrane and Mtiller-Eberhard, 1968); and determination by amino acid analysis technique. This last procedure involved passing samples over a column of Beckman AA-20 ion exchange resin. A buffer of 0.35 M Na ÷ (as sodium citrate) at pH 5.28 was used to elute extraneous material from the column, then at 20 min into the run a Kobayashi buffer (Kobayashi, 1980) at pH 9.75 was applied to elute the histamine from the column. All eluates were analyzed by a Beckman 121-M amino acid analyzer. Determination of protein content The total protein content of 9 of the samples was determined by Folin assay a n d / o r by amino acid analysis (vide infra). Determination of amino acid composition This method employed a Beckman 121-M amino acid analyzer using a technique described previously (Fernandez and Hugli, 1976).
Results
The sensitivities o f the 3 methods used for histamine determination are reflected by the following lower limits of detectability: fluorometric determination, 1 n g / m l ; guinea pig ileum, 13 ng; and amino acid analysis technique, 500 ng/ml. Significant quantities of histamine were found in all commercial samples, ranging from 0.026 # g / m l (10 -7 M) to 167.5 # g / m l (10 3 M) (Table I). Results of the fluorometric and amino acid analyses were similar. The bioassay consistently showed 3-5 times less histamine than did amino acid analysis. This overall consistency is remarkable in
357 TABLE I HISTAMINE CONTENT OF PWM SAMPLES (/tg/ml) Sample (source)
Fluorometric
Guinea pig ileum
Amino acid analysis
1A 1B 1C 1D 2 3A 3B 3C 4 5
> 10 > 10 > 10 > 10 5.0 1.5 N.D. N.D. 0.125 0.150
26.0 60.0 25.1 N.D. a N.D. N.D. 1.55 1.00 0.026 N.D.
91.5 160.0 77.5 167.5 3.1 1.0 <5 <5 <5 <5
a N.D., not done.
view of the t r e m e n d o u s variability in h i s t a m i n e d e t e r m i n a t i o n that has been r e p o r t e d ( G l e i c h and Hull, 1980). In fact, a u t o m a t e d f l u o r o m e t r y was f o u n d to be o n e of the m o s t reliable m e t h o d s e x a m i n e d for buffered samples. T a b l e II depicts the total p r o t e i n c o n t e n t of those P W M samples assayed. Values r a n g e d f r o m 0.56 to 4.4 mg. This variation was observed in samples f r o m different c o m p a n i e s (differing by as m u c h as 550%), a n d to a lesser degree a m o n g different lots from the same c o m p a n y (differing by as m u c h as 30%). T h e a m i n o acid c o m p o s i t i o n s of P W M in the 5 samples tested are shown in T a b l e III. This pattern, c o m p o s e d largely of aspartic acid, glutamic acid, glycine, and half-cystine, is characteristic of P W M (Reisfeld et al., 1967; Waxdal, 1974; Y o k o y a m a
TABLE II TOTAL PROTEIN CONTENT OF PWM SAMPLES (mg) Sample (source)
Folin
Amino acid analysis
IA IB IC 1D 2 3A 3B 3C 4
N.D. a 0.80 0.60 N.D. 1.25 4.00 4.40 4.20 1.90
N.D. N.D. N.D. 0.56 2.21 3.64 N.D. N.D. 2.13
5
1.30
1.40
a N.D., not done.
500 400 400 1 130 500 690 2020 720 1400 850 N.D. b 610 80 270 530 < 40 270
4.4 3.5 3.5 9.9 4.4 6.1 17.8 6.3 12.3 7.5 N.D. 5.4 0.7 2.4 4.7 < 0.35 2.4
1 260 510 940 2 670 1 100 1 610 2330 970 2 620 1 340 1 960 a 1 360 80 760 1 320 820 880
n moles/ml
n moles/ml
%
Source 2
Source 1D
5.6 2.3 4.2 11.9 4.9 7.1 10.3 4.3 11.6 5.9 8.7 a 6.0 0.4 3.4 5.9 3.6 3.9
% 1 690 870 1 850 4 990 1 710 2600 4730 1 620 5 940 1 380 N.D. b 1 480 300 530 1 580 1 590 1 060
n moles/ml
Source 3C
4.5 2.5 5.0 13.4 4.6 7.0 12.7 4.4 16.0 3.7 N.D. 4.0 0.8 1.4 4.3 4.3 2.9
% 1 260 530 710 2820 1 10f) 1 500 2220 1 540 2790 1 110 N.D. b 1 270 120 630 1 300 820 380
n moles/ml
Source 4
5.7 2.4 3.2 12.8 5.0 6.8 10.7 7.0 12.7 5.0 N.D. 5.8 0.5 2.9 5.9 3.7 1.7
%
870 370 460 1 940 780 1 030 750 1 080 1 940 770 N.D. b 900 80 430 890 650 350
n moles/ml
Source 5
6.0 2.5 3.2 13.3 5.4 7.1 5.2 7.4 13.3 5.3 N.D. 6.2 0.5 3.0 6.1 4.5 2.4
%
a Half-cystine p r o p o r t i o n measured for source 2 only and a s s u m e d for all other samples. b Total molar concentration for s a m p l e adjusted to include 8.7% for half cystine; N.D., not done. T r y p t o p h a n not measured, a s s u m e d to be - 2% and excluded from calculations in all samples.
Lysine Histidine Arginine Aspartic acid Threonine Serine G l u t a m i c acid Proline Glycine Alanine H a l f cystine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine
A m i n o acid
AMINO ACID COMPOSITION OF FIVE PWM PREPARATIONS
TABLE Ill
359 et al., 1976). However, the proportions of the 3 amino acid residues: glutamic acid, glycine, and alanine varied significantly from one sample to the next.
Discussion
The finding of large quantities of histamine in commercial PWM preparations may have significance in understanding reported variations in in vitro lymphocyte responses to this mitogen. While PWM stimulated the synthesis of IgG, IgM, and IgA in human mononuclear cells (Waldmann et al., 1974), studies of PWM-induced IgE synthesis have yielded conflicting data. Some studies (Saxon and Stevens, 1979; Saxon et al., 1980a; Pryjma et al., 1980) provide evidence that PWM can stimulate IgE synthesis, while other investigations (Buckley and Becker, 1978; Saxon et al., 1980b; Buckley et al., 1981) suggest that PWM may actually inhibit the production of IgE. As little as 1 0 - 6 M histamine has been reported to activate suppressor T lymphocytes in Con A and PHA human mononuclear cell systems (Rocklin and Haberek-Davidson, 1981). From analysis of our data, the final concentration of histamine in human cell cultures of IgE synthesis studies would be approximately 10 - 6 M. A 1 : 100 dilution (w/v) of Gibco PWM was used in all studies cited. This quantity of histamine could potentially activate T suppressor cells in PWM-stimulated human mononuclear cell systems. Consequently, the variance in histamine contamination of PWM preparations could account for some of the discrepancies reported in the studies of PWM induction of IgE synthesis. Some preparations might contain insignificant amounts of histamine, while others might possess quantities sufficient to suppress IgE synthesis. The variability in protein content for a putative quantity of commercial PWM suggests that the dose calculated (based upon the manufacturer's specifications) may differ from the dose actually used. This could be important in a cell culture system with a narrow dose-response curve (Janossy and Greaves, 1971). The variability in amino acid composition could also affect the ability of PWM to stimulate immunoglobulin synthesis. These disparities offer alternative explanations for the confusion regarding the ability of PWM to stimulate de novo IgE synthesis. Commercial producers often control for these discrepancies by adjusting the dose of PWM employed from a given lot to yield maximal biological activity. Unfortunately, these functional assays are not always the same as those of interest to the immunologist, e.g., proliferation or immunoglobulin synthesis. Moreover, one cannot assume dose-response equivalency for these different phenomena. The only way to control optimally for these variations is to obtain dose-response data for each lot, measuring the parameters one intends to assess. In summary, we have demonstrated large quantities of histamine, a potent biological substance, in 10 samples of PWM from 5 commercial sources. Not only was histamine detected using the highly sensitive and specific isotopic enzymatic and fluorometric assays, but its presence was also identified by an amino acid analysis technique and confirmed by bioassay. We also report fairly wide variability in the
360 p r o t e i n c o n t e n t o f t h e s e s a m p l e s . F i n a l l y , d i f f e r e n c e s in 3 o f t h e a m i n o a c i d r e s i d u e s of the protein preparations were found. The functional significance of our findings r e q u i r e s f u r t h e r s t u d y . H o w e v e r , p r e v i o u s s t u d i e s in w h i c h P W M w a s u s e d to i n d u c e m o n o n u c l e a r cell p r o l i f e r a t i o n o r i m m u n o g l o b u l i n t a t i o n in v i e w o f t h e s e results.
synthesis may require reinterpre-
References Buckley, R.H. and W.G. Becker, 1978, lmmunol. Rev. 41,288. Buckley, R.H., H.A. Sampson and P.M. Fiser, 1981, in: Diagnosis and Treatment of IgE-mediated Diseases, ed. S.G.O. Johansson (Excerpta Medica, New York) p. 6. Cochrane, C.G. and H.J. Mfiller-Eberhard, 1968, J. Exp. Med. 127, 371. Douglas, S.D., 1972, Transplant. Rev. 11, 39. Fernandez, H.N. and T.E. Hugli, 1976, J. Immunol. 117, 1688. Gleich, G.J. and W.M. Hull, 1980, J. Allergy Clin. Immunol. 66, 295. Janossy, G. and M.F. Greaves, 1971, Clin. Exp. Immunol. 9, 483. Janossy, G. and M.F. Greaves, 1972, Clin. Exp. Immunol. 10, 525. Kobayashi, Y., 1980, Anal. Biochem. 105, 48. O'Connor, R.D., J. Ring, A.A. Jalowayski and R.N. Hamburger, 1980, Clin. Allergy 10, 705. Pryjma, J., J. Munoz, G. Virella and H.H. Fudenberg, 1980, Cell Immunol. 50, 115. Reisfeld, R.A., J. B~rjeson, L.N. Chessin and P.A. Small, Jr., 1967, Proc. Natl. Acad. Sci. U.S.A. 58, 2020. Rocklin, R.E. and A. Haberek-Davidson, 1981, J. Clin. Immunol. 1, 73. Sampson, H.A. and R.H. Buckley, 1981, J. Immunol. 127, 829. Saxon, A. and R.H. Stevens, 1979, Clin. Immunol. Immunopathol. 14, 474. Saxon, A., M. Kaplan and R.H. Stevens, 1980a, J. Allergy Clin. Immunol. 66, 233. Saxon, A., C. Morrow and R.H. Stevens, 1980b, J. Clin. Invest. 65, 1457. Siraganian, R.P., 1974, Anal. Biochem. 57, 383. Waldmann, T.A., S. Broder, R.M. Blaese, M. Durm, M. Blackman and W. Strober, 1974, Lancet ii, 609. Wang, S.R. and B. Zweiman, 1978, Cell. Immunol. 36, 28. Waxdal, M.J., 1974, Biochemistry 13, 3671. Yokoyama, K., O. Yano, T. Terao and T. Osawa, 1976, Biochim. Biophys. Acta 427, 443.
355
Elsevier Biomedical Press
Histamine Contamination of Pokeweed Mitogen James M. Seltzer, Richard D. O'Connor 1, Ronald A. Simon and Tony E. Hugli Immunology and Allergy Division, Department of Pediatrics, School of Medicine, University of California, San Diego, La Jolla, CA 92093, and Division of Allergy and Clinical Immunology, and Department of Molecular Immunology, Scrzpps Clinic and Research Foundation, La Jolla, CA 92037, U.S.A.
(Received 26 May 1982, accepted 18 June 1982)
The histamine content of commercial preparations of pokeweed mitogen was measured by amino acid analysis technique, automated fluorometry, and bioassay employing the guinea pig ileum. Ten samples from 5 companies were examined and found to contain between 0.026 #g and 167.5 #g of histamine per ml of solution. The protein content of 9 of these putative 5 mg samples measured by folin assay and by amino acid analysis varied from 0.56 to 4.4 mg. Their amino acid compositions were similar, except for notable variations in 3 of the 16 residues quantitated. Key words: pokeweed mitogen - - histamine - - lgE
Introduction P o k e w e e d m i t o g e n ( P W M ) is an extract of the weed P h y t o l a c c a A m e r i c a n a that stimulates b o t h B a n d T l y m p h o c y t e s in vitro (Douglas, 1972; J a n o s s y a n d Greaves, 1972). This s u b s t a n c e has b e e n used p r i m a r i l y to s t u d y in vitro m o n o n u c l e a r cell p r o l i f e r a t i o n a n d i m m u n o g l o b u l i n (Ig) synthesis. Samples available from c o m m e r c i a l sources are p u r i f i e d b y a variety of different techniques. R e c e n t studies have shown that h i s t a m i n e inhibits m o n o n u c l e a r cell p r o l i f e r a t i o n i n d u c e d b y the p l a n t m i t o g e n s c o n c a n a v a l i n A ( C o n A) a n d p h y t o h e m a g g l u t i n i n ( P H A ) . This effect a p p e a r s to be m e d i a t e d b y l y m p h o c y t e s b e a r i n g h i s t a m i n e type 2 r e c e p t o r s on their surface ( W a n g and Z w e i m a n , 1978; R o c k l i n a n d H a b e r e k - D a v i d son, 1981). M o n o n u c l e a r cells f r o m a t o p i c p a t i e n t s with extreme elevations of IgE have d e m o n s t r a t e d p o o r proliferative responses to P W M ( O ' C o n n o r et al., 1980). Since these results were o b s e r v e d using a whole b l o o d culture technique that preserves b a s o p h i l function, we speculated that P W M might serve as an allergen causing I g E - m e d i a t e d release of histamine. T o test this p o s s i b i l i t y the h i s t a m i n e
1Address reprint requests to Richard D. O'Connor at U.C.S.D., School of Medicine, Immunology and Allergy Division, Department of Pediatrics (M-009-D), La Jolla, CA 92093, U.S.A. 0022-1759/82/0000-0000/$02.75
© Elsevier Biomedical Press
356
concentration of PWM-stimulated cell culture supernatants was measured. Large amounts of histamine, determined by radioisotopic enzymatic assay, were found in supernatant fluids from cell cultures as well as in cell-free control samples which contained only PWM. We then examined the histamine content of PWM samples available from several commercial sources to determine if PWM could be the source of histamine.
Materials and Methods
Preparation and storage of P WM Ten 5 mg samples of PWM obtained from 5 companies were analyzed for the presence of histamine. Each sample represents a different lot number. All samples were diluted in 1 ml sterile phosphate-buffered saline, and stored at - 2 0 ° C until assayed. Determination of h&tamine content Three methods were used to document reliability of our results: automated fluorometry modified from Siraganian (1974); histamine-induced contraction of guinea pig ileum smooth muscle (Cochrane and Mtiller-Eberhard, 1968); and determination by amino acid analysis technique. This last procedure involved passing samples over a column of Beckman AA-20 ion exchange resin. A buffer of 0.35 M Na ÷ (as sodium citrate) at pH 5.28 was used to elute extraneous material from the column, then at 20 min into the run a Kobayashi buffer (Kobayashi, 1980) at pH 9.75 was applied to elute the histamine from the column. All eluates were analyzed by a Beckman 121-M amino acid analyzer. Determination of protein content The total protein content of 9 of the samples was determined by Folin assay a n d / o r by amino acid analysis (vide infra). Determination of amino acid composition This method employed a Beckman 121-M amino acid analyzer using a technique described previously (Fernandez and Hugli, 1976).
Results
The sensitivities o f the 3 methods used for histamine determination are reflected by the following lower limits of detectability: fluorometric determination, 1 n g / m l ; guinea pig ileum, 13 ng; and amino acid analysis technique, 500 ng/ml. Significant quantities of histamine were found in all commercial samples, ranging from 0.026 # g / m l (10 -7 M) to 167.5 # g / m l (10 3 M) (Table I). Results of the fluorometric and amino acid analyses were similar. The bioassay consistently showed 3-5 times less histamine than did amino acid analysis. This overall consistency is remarkable in
357 TABLE I HISTAMINE CONTENT OF PWM SAMPLES (/tg/ml) Sample (source)
Fluorometric
Guinea pig ileum
Amino acid analysis
1A 1B 1C 1D 2 3A 3B 3C 4 5
> 10 > 10 > 10 > 10 5.0 1.5 N.D. N.D. 0.125 0.150
26.0 60.0 25.1 N.D. a N.D. N.D. 1.55 1.00 0.026 N.D.
91.5 160.0 77.5 167.5 3.1 1.0 <5 <5 <5 <5
a N.D., not done.
view of the t r e m e n d o u s variability in h i s t a m i n e d e t e r m i n a t i o n that has been r e p o r t e d ( G l e i c h and Hull, 1980). In fact, a u t o m a t e d f l u o r o m e t r y was f o u n d to be o n e of the m o s t reliable m e t h o d s e x a m i n e d for buffered samples. T a b l e II depicts the total p r o t e i n c o n t e n t of those P W M samples assayed. Values r a n g e d f r o m 0.56 to 4.4 mg. This variation was observed in samples f r o m different c o m p a n i e s (differing by as m u c h as 550%), a n d to a lesser degree a m o n g different lots from the same c o m p a n y (differing by as m u c h as 30%). T h e a m i n o acid c o m p o s i t i o n s of P W M in the 5 samples tested are shown in T a b l e III. This pattern, c o m p o s e d largely of aspartic acid, glutamic acid, glycine, and half-cystine, is characteristic of P W M (Reisfeld et al., 1967; Waxdal, 1974; Y o k o y a m a
TABLE II TOTAL PROTEIN CONTENT OF PWM SAMPLES (mg) Sample (source)
Folin
Amino acid analysis
IA IB IC 1D 2 3A 3B 3C 4
N.D. a 0.80 0.60 N.D. 1.25 4.00 4.40 4.20 1.90
N.D. N.D. N.D. 0.56 2.21 3.64 N.D. N.D. 2.13
5
1.30
1.40
a N.D., not done.
500 400 400 1 130 500 690 2020 720 1400 850 N.D. b 610 80 270 530 < 40 270
4.4 3.5 3.5 9.9 4.4 6.1 17.8 6.3 12.3 7.5 N.D. 5.4 0.7 2.4 4.7 < 0.35 2.4
1 260 510 940 2 670 1 100 1 610 2330 970 2 620 1 340 1 960 a 1 360 80 760 1 320 820 880
n moles/ml
n moles/ml
%
Source 2
Source 1D
5.6 2.3 4.2 11.9 4.9 7.1 10.3 4.3 11.6 5.9 8.7 a 6.0 0.4 3.4 5.9 3.6 3.9
% 1 690 870 1 850 4 990 1 710 2600 4730 1 620 5 940 1 380 N.D. b 1 480 300 530 1 580 1 590 1 060
n moles/ml
Source 3C
4.5 2.5 5.0 13.4 4.6 7.0 12.7 4.4 16.0 3.7 N.D. 4.0 0.8 1.4 4.3 4.3 2.9
% 1 260 530 710 2820 1 10f) 1 500 2220 1 540 2790 1 110 N.D. b 1 270 120 630 1 300 820 380
n moles/ml
Source 4
5.7 2.4 3.2 12.8 5.0 6.8 10.7 7.0 12.7 5.0 N.D. 5.8 0.5 2.9 5.9 3.7 1.7
%
870 370 460 1 940 780 1 030 750 1 080 1 940 770 N.D. b 900 80 430 890 650 350
n moles/ml
Source 5
6.0 2.5 3.2 13.3 5.4 7.1 5.2 7.4 13.3 5.3 N.D. 6.2 0.5 3.0 6.1 4.5 2.4
%
a Half-cystine p r o p o r t i o n measured for source 2 only and a s s u m e d for all other samples. b Total molar concentration for s a m p l e adjusted to include 8.7% for half cystine; N.D., not done. T r y p t o p h a n not measured, a s s u m e d to be - 2% and excluded from calculations in all samples.
Lysine Histidine Arginine Aspartic acid Threonine Serine G l u t a m i c acid Proline Glycine Alanine H a l f cystine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine
A m i n o acid
AMINO ACID COMPOSITION OF FIVE PWM PREPARATIONS
TABLE Ill
359 et al., 1976). However, the proportions of the 3 amino acid residues: glutamic acid, glycine, and alanine varied significantly from one sample to the next.
Discussion
The finding of large quantities of histamine in commercial PWM preparations may have significance in understanding reported variations in in vitro lymphocyte responses to this mitogen. While PWM stimulated the synthesis of IgG, IgM, and IgA in human mononuclear cells (Waldmann et al., 1974), studies of PWM-induced IgE synthesis have yielded conflicting data. Some studies (Saxon and Stevens, 1979; Saxon et al., 1980a; Pryjma et al., 1980) provide evidence that PWM can stimulate IgE synthesis, while other investigations (Buckley and Becker, 1978; Saxon et al., 1980b; Buckley et al., 1981) suggest that PWM may actually inhibit the production of IgE. As little as 1 0 - 6 M histamine has been reported to activate suppressor T lymphocytes in Con A and PHA human mononuclear cell systems (Rocklin and Haberek-Davidson, 1981). From analysis of our data, the final concentration of histamine in human cell cultures of IgE synthesis studies would be approximately 10 - 6 M. A 1 : 100 dilution (w/v) of Gibco PWM was used in all studies cited. This quantity of histamine could potentially activate T suppressor cells in PWM-stimulated human mononuclear cell systems. Consequently, the variance in histamine contamination of PWM preparations could account for some of the discrepancies reported in the studies of PWM induction of IgE synthesis. Some preparations might contain insignificant amounts of histamine, while others might possess quantities sufficient to suppress IgE synthesis. The variability in protein content for a putative quantity of commercial PWM suggests that the dose calculated (based upon the manufacturer's specifications) may differ from the dose actually used. This could be important in a cell culture system with a narrow dose-response curve (Janossy and Greaves, 1971). The variability in amino acid composition could also affect the ability of PWM to stimulate immunoglobulin synthesis. These disparities offer alternative explanations for the confusion regarding the ability of PWM to stimulate de novo IgE synthesis. Commercial producers often control for these discrepancies by adjusting the dose of PWM employed from a given lot to yield maximal biological activity. Unfortunately, these functional assays are not always the same as those of interest to the immunologist, e.g., proliferation or immunoglobulin synthesis. Moreover, one cannot assume dose-response equivalency for these different phenomena. The only way to control optimally for these variations is to obtain dose-response data for each lot, measuring the parameters one intends to assess. In summary, we have demonstrated large quantities of histamine, a potent biological substance, in 10 samples of PWM from 5 commercial sources. Not only was histamine detected using the highly sensitive and specific isotopic enzymatic and fluorometric assays, but its presence was also identified by an amino acid analysis technique and confirmed by bioassay. We also report fairly wide variability in the
360 p r o t e i n c o n t e n t o f t h e s e s a m p l e s . F i n a l l y , d i f f e r e n c e s in 3 o f t h e a m i n o a c i d r e s i d u e s of the protein preparations were found. The functional significance of our findings r e q u i r e s f u r t h e r s t u d y . H o w e v e r , p r e v i o u s s t u d i e s in w h i c h P W M w a s u s e d to i n d u c e m o n o n u c l e a r cell p r o l i f e r a t i o n o r i m m u n o g l o b u l i n t a t i o n in v i e w o f t h e s e results.
synthesis may require reinterpre-
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