The potentially insecticidal Narcissus pseudonarcissus lectindemonstrates age-related mitogenicity

Christina Summers a, John Forrest b, Mary Norval c, James Michael Sharp a;�

a Moredun Research Institute, Pentland Science Park, Bush Loan, Penicuik, Midlothian EH26 0PZ, UKb Scottish Crop Research Institute, Invergowrie, Dundee, UK

c Medical Microbiology, University of Edinburgh, Medical School, Treviot Place, Edinburgh, UK

Received 18 December 2001; accepted 19 December 2001

First published online 4 February 2002

Abstract

Lectins from monocotyledonous plants such as Narcissus pseudonarcissus (NPA) possess insecticidal properties and have the potentialto increase pest resistance in transgenic crops. Therefore it is of interest to investigate the mitogenic properties of such lectins.Mononuclear cells purified from human umbilical cord and adult peripheral blood samples were stimulated with NPA and compared tophytohaemagglutinin as an example of a lectin from a dicotyledonous plant. Here we report that NPA is slightly mitogenic for adulthuman lymphocytes but mitogenicity is increased more than sevenfold for lymphocytes from umbilical cord blood. Similarly, NPA wasfound to be mitogenic for peripheral blood mononuclear cells (PBMC) from lambs and not adult sheep, supporting the age-relatedmitogenicity and indicating that further examination of the younger human population is warranted. ß 2002 Federation of EuropeanMicrobiological Societies. Published by Elsevier Science B.V. All rights reserved.

Keywords: Human lymphoproliferation; Mitogenicity; Narcissus pseudonarcissus lectin

1. Introduction

Plant lectins have been used as mitogens for severaldecades to evaluate immunocompetence. Most commer-cially available lectins are obtained from dicotyledonousplants, including phytohaemagglutinin (PHA) from the redkidney bean, Phaseolus vulgaris. An increased interest inmonocotyledonous lectins has developed following the dis-covery that some lectins of the super-family Amaryllida-ceae, such as snowdrop Galanthus nivalus (GNA) and daf-fodil Narcissus pseudonarcissus (NPA), possess insecticidalproperties. Several prototype arable crop plants are nowtransgenic for GNA to enable preliminary assessment oftheir pest resistance [1,2]. Lectins have wide biological ac-tivity and concerns have been expressed about consump-tion of, or exposure to, crops transgenic for lectins. Thefew studies examining human subjects found GNA andNPA to be virtually non-mitogenic [3,4]. In one instancethese preliminary reassuring observations were obtained

with lymphocytes from adult donors, all over the age of60 [4]. But we now report that the mitogenicity of NPA isage-related, with the lymphoproliferative response of theperipheral blood mononuclear cells (PBMC) in umbilicalcord blood higher than in adult blood. Similarly, NPAwas found to be mitogenic for PBMC from lambs andnot adult sheep, supporting the age-related mitogenicityand indicating that further examination of the youngerhuman population is warranted.

2. Materials and methods

2.1. Human subjects

In total, 12 human blood samples were collected, 7 um-bilical cord bloods obtained during routine caesarean sec-tions (kindly collected by Dr R. Hughes, Royal In¢rmaryof Edinburgh, following informed consent) and 5 volun-teer adults, ages ranging from 24 to 56 years.

2.2. Sheep

In total, PBMC from 74 blood samples were assayed

0928-8244 / 02 / $22.00 ß 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.PII: S 0 9 2 8 - 8 2 4 4 ( 0 2 ) 0 0 2 7 1 - 7

* Corresponding author.Tel. : +44 (131) 445 5111; Fax: +44 (131) 445 6111.

E-mail address: sharm@mri.sari.ac.uk (J. Michael Sharp).

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and analysed in three groups: lambs aged 4 days^1 month (Group A, n = 37), 2^4 months (Group B,n = 26), and adult sheep aged 1^3 years (Group 3,n = 11). All animals were from the Moredun Research In-stitute.

2.3. Blood samples

Human umbilical cord and adult venous blood sampleswere collected into sterile tubes containing 10 units pres-ervative free heparin per ml blood. The mononuclear cellswere puri¢ed and adjusted to a ¢nal concentration of 106

cells ml31 in RPMI-1640 medium (Life Technologies Ltd)containing antibiotics and 10% autologous plasma (humansamples) or 10% heat inactivated foetal calf serum (sheepsamples).

2.4. Cell culture with lectins

The optimum quantities, determined from a standardcurve, of commercially purchased PHA (Sigma, Poole,Dorset, UK) and NPA (Vector Laboratories, Peterbor-ough, UK) were added to 106 PBMC ml31. Control sam-ples (excluding lectin) were also prepared. All samples, at2U105 cells/well were dispensed, (human samples ¢ve rep-licates, sheep samples in triplicate) into 96-well microtitreplates. The lymphocyte proliferation assays were per-formed in accordance with standard laboratory protocol[5] for a total of 96 h, with the addition of 0.7 WCi ml31

[methyl-3H]thymidine (speci¢c activity 2.0 Ci/mmol, Amer-sham, Little Chalfont, Buckinghamshire, UK) for the ¢nal16 h. The cells were harvested and [3H]thymidine uptakemeasured as cpm. For each sample the mean cpm of the

Fig. 1. In vitro lymphoproliferative response of PBMC to NPA (E) and PHA (F) stimulation. [3H]thymidine uptake, shown as cpm þ S.E.M. A; Humanresponses for umbilical cord blood (UCB) and adult blood. Comparison between the two groups was not statistically signi¢cant for PHA stimulation,but highly signi¢cant (P = 0.0004) for NPA. B: Ovine responses for group A lambs aged 4 days^1 month, group B lambs aged 2^4 months and groupC adult sheep aged 1^3 years. Comparison between the two groups of lambs (A, B) and the adults (group C) were not statistically signi¢cant for PHAstimulation, but were signi¢cant for NPA, at P = 0.0005 for group A and P = 0.04 for group B.

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replicates was calculated and the control mean cpm sub-tracted: S.E.M. was determined. Two-tailed student t-testdetermined the level of signi¢cance between the humanadult and umbilical cord mononuclear cells, and betweenadult sheep and the two groups of lambs.

3. Results

PHA was strongly mitogenic for lymphocytes of bothhuman age groups (umbilical cord mononuclear cellsmean cpm 65 058 þ 12 108 and adult PBMC76 161 þ 13 770) and there was no statistically signi¢cantdi¡erence in these responses (Fig. 1A). In contrast, theproliferative response to NPA stimulation was signi¢-cantly higher (P = 0.0004) with umbilical cord mononu-clear cells (mean cpm 11 322 þ 1549) compared to adultPBMC (mean cpm 1580 þ 413) (Fig. 1A).

In sheep, no statistically signi¢cant di¡erence in thelymphocyte stimulation was found between the age groupsin response to PHA (Fig. 1B). Stimulation with NPA gavea mean cpm of 5154 þ 445 and 2203 þ 616 for groups Aand B respectively, compared to 330 þ 128 for the adultgroup C (Fig. 1B). The statistical signi¢cance of theseresults, in comparison to the adult group C, was calculatedat P = 0.0005 for group A and P = 0.04 for group B.

4. Discussion

This study has provided clear evidence, in contrast toprevious reports [4,5] that the monocotyledonous lectin,NPA, is mitogenic for human lymphocytes. We have con-¢rmed that the proliferative response to NPA stimulationis age-related, with weak mitogenicity identi¢ed for adulthuman lymphocytes, and increasing more than sevenfoldfor lymphocytes from umbilical cord blood. Likewise,NPA was found to be mitogenic for PBMC from lambs

and not adult sheep. As no age-dependent response toPHA stimulation was evident in either humans or sheep,and the lymphoproliferative responses to NPA in sheepre£ected our observations in humans, the results presentedhere suggest that age-related divergence in the mononu-clear cell population a¡ects the mitogenicity of NPA. Our¢ndings relate only to in vitro studies and cannot predictin vivo responses to NPA. However, these results indicatethat exposure of the human population to NPA couldhave physiological consequences. Therefore, to appreciatefully the potential implications of introducing foreign lec-tins into human and animal food sources, it is essentialthat we extend our knowledge of the plant lectin^mamma-lian cell relationship, not only for species speci¢city but toinclude all age groups.

Acknowledgements

The authors wish to express their gratitude to Dr RhonaHughes, Royal In¢rmary of Edinburgh. This work wasfunded by Scottish Executive, Environmental and RuralA¡airs Department.

References

[1] Down, R.E., Gatehouse, A.M.R., Hamilton, W.D.O. and Gatehouse,J.A. (1996) J. Insect Physiol. 42, 1035^1045.

[2] Machuka, J., Van Damme, E.J.M. and Peumans, W.J. (1999) Ento-mol. Exp. Appl. 93, 179^187.

[3] Kilpatrick, D.C., Peumans, W.J. and Van Damme, E.J.M. (1990)Clin. Biochem. 7, 259^263.

[4] Fenton, B., Stanley, K., Fenton, S. and Bolton-Smith, C. (1999)Di¡erential binding of the insecticidal lectin GNA to human bloodcells. Lancet 354, 1354^1355.

[5] Kilpatrick, D.C. (1998). Methods in Molecular Medicine, Vol. 9:Lectin Methods and Protocols (Rhodes, J.M. and Milton J.D.,Eds.), Humana, Totowa, NJ.

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