In two in vitro studies, we examined the immunological (pathways of the) effects of Citrus/Cydonia comp. from, respectively, a healthy and an allergic donor; peripheral blood mononuclear cells (PBMCs) were isolated out of peripheral blood and analyzed in vitro after polyclonal stimulation of T-cells. The differentiation capacity and the influence with regard to Th1 (IFN-
Allergic rhinitis is a condition characterized by sneezing, watery nasal
discharge, and nasal obstruction and itching. It is an increasingly prevalent
condition, particularly in the Western world where it affects around 20% of the
adult population. Allergic rhinitis is divided into seasonal allergic rhinitis
(hay fever) which is triggered by pollens and moulds, and perennial allergic
rhinitis in which house dust mites and pet dander are the predominant triggers.
The spectrum of severity is wide and includes a significant number of sufferers
with severe symptoms that are resistant to treatment with usual pharmacotherapy
(antihistamines and topical nasal corticosteroids) [
Seasonal allergic
rhinitis or hay fever is a type I immediate hypersensitivity reaction
mediated by specific IgE antibody to a seasonal allergen, leading to
mucosal inflammation characterized by sneezing, itching, rhinorrhoea,
and nasal blockage. Pollens (6–40
Citrus/Cydonia
comp. is an anthroposophic medicine, which contains extracts of lemon (Citrus
lemon) and quince (Cydonia oblongata) [
From a healthy and an allergic individual, 8 mL of blood was collected in sodium heparinate-coated vacutainers (BD Biosciences, San Diego, Calif, USA). The allergic individual was sensitized for birch pollen (RAST 6+) and grass pollen (RAST 4+), and he was also food allergic with a positive skin prick test on apple and cherry. The blood was subsequently diluted 1:1 with IMDM containing GlutaMAX (IMDM; Gibco-BRL, Paisley, Scotland) before the density gradient centrifugation on Ficoll-Paque PLUS (Amersham Biosciences, Uppsala, Sweden). The PBMC layer was washed twice with IMDM and the cell viability and cell concentration were determined by Trypan blue exclusion. An informed consent was obtained before the sample collection and the performed experiments were approved by the local ethical committee.
PBMCs were cultured in Yssel’s
medium at
Both conditions (negative and
positive controls) took place in the presence of Citrus/Cydonia comp. 100
Half a million PBMCs were
washed and subsequently incubated with 2
The
immunological phenotype of PBMC subsets was determined by staining the surface
antigens with the following two monoclonal antibody (
Per well,
The proliferation capacity of
the PBMC was studied by intracellular expression of the nuclear Ki-67 antigen
(Ki-67; BD Pharmingen). The Ki-67 antigen is absent in the nuclei of resting cells, but
present in all other phases of the cell division cycle as well as in the
mitosis phase [
PBMC culture supernatants were
analyzed for their IL-1
PBMCS were isolated and analyzed
for their subset composition. The percentages of the subsets PBMC were 63% CD3+
T-cells (with 50.4% CD4+ Th-cells and 12.6% CD8+ Tc-cells), 8% CD19+ B cells,
5% CD14+ monocytes, and 12% CD16/CD56+ NK cells. After one day (Table
Mean scores after one day (healthy donor).
Medium | Medium + Citrus/Cydonia comp. 1 | Medium + Citrus/Cydonia comp. 1:3 | |
---|---|---|---|
Proliferation (%) | 3
| 13 (3) | 1 (1) |
Cell death (%) | 88 (12) | 82 (11) | 87 (5) |
Cytokines (pg/mL) | |||
TNF- | 13 (4) | 3315 (129) | 13 (1) |
IL-1 | 25 (6) | 3535 (147) | 15 (2) |
IL-10 | 22 (9) | 918 (52) | 12 (1) |
IL-12 | 12 (3) | 46 (12) | 12 (2) |
IFN- | 15 (2) | 55 (8) | 25 (3) |
IL-4 | 10 (2) | 12 (1) | 10 (1) |
IL-5 | 12 (2) | 28 (3) | 10 (1) |
Percentages of proliferating cells and cells in apoptosis of PBMC cultures of a healthy donor stimulated for one day with medium or Citrus/Cydonia undiluted and 1:3 diluted. In the supernatants of these cultures, cytokines were measured by flow cytometric analysis in the Bead Assay Flex Sets system. Cytokine levels in pg/mL. *All results are described with standard deviations (SDs).
Mean scores after four days (healthy donor).
Medium | Anti-CD3/28 | Medium + Citrus/Cydonia comp. 1 | Medium + Citrus/Cydonia comp. 1:3 | Stimulation + Citrus/Cydonia comp. 1 | Stimulation + Citrus/Cydonia comp. 1:3 | |
---|---|---|---|---|---|---|
Proliferation (%) | 3
| 42 (8) | 3 (1) | 1 (1) | 48 (11) | 40 (2) |
Cell death (%) | 88 (12) | 61 (11) | 87 (8) | 92 (11) | 65 (12) | 60 (6) |
Cytokines (pg/mL) | ||||||
TNF- | 13 (2) | 8483 (987) | 15 (2) | 13 (2) | 9647 (733) | 8117 (566) |
IL-1 | 15 (3) | 4134 | 35 (11) | 15 (1) | 4858 (247) | 4037 (138) |
IL-10 | 12 (2) | 9553 | 18 (8) | 12 (2) | 12276 (566) | 5662 (931) |
IL-12 | 12 (1) | 84 | 26 (9) | 12 (2) | 238 (87) | 185 (77) |
IFN- | 15 (2) | 34355 | 15 (1) | 15 (2) | 48800 (12778) | 37750 (12501) |
IL-4 | 12 (2) | 134 | 12 (1) | 12 (2) | 55 (12) | 106 (28) |
IL-5 | 12 (2) | 375 | 12 (2) | 12 (1) | 118 (45) | 266 (56) |
Percentages of proliferating cells and cells in apoptosis of PBMC cultures of a healthy donor stimulated for four days with medium alone, polyclonal stimulation with anti-CD3 plus anti-CD28 antibodies, or polyclonal stimulation in the presence of Citrus/Cydonia preparation undiluted and 1:3 diluted. In the supernatants of these cultures, cytokines were measured by flow cytometric analysis in the Bead Assay Flex Sets system. Cytokine levels in pg/mL. *All results are described with standard deviations (SDs).
After four days (Table
Mean scores after four days (allergic donor).
Medium | Stimulation with anti-CD3/28 | Stimulation + Citrus/Cydonia comp. | |
---|---|---|---|
Proliferation (%) | 1
| 48 (12) | 57 (12) |
Cell death (%) | 78 (8) | 59 (16) | 55 (8) |
Cytokines | |||
TNF- | 10 (1) | 5782 (154) | 6893 (738) |
IL-1 | 10 (1) | 2275 (339) | 3772 (665) |
IL-10 | 10 (1) | 2331 (452) | 7634 (1299) |
IL-12 | 10 (1) | 134 (26) | 335 (89) |
IFN- | 10 (1) | 9778 (452) | 11668 (1638) |
IL-4 | 10 (1) | 456 (87) | 138 (18) |
IL-5 | 10 (1) | 667 (154) | 227 (85) |
Percentages of proliferating cells and cells in apoptosis of PBMC cultures of an allergic donor stimulated for four days with medium alone, polyclonal stimulation with anti-CD3 plus anti-CD28 antibodies, or in the presence of Citrus/Cydonia undiluted preparation. In the supernatants of these cultures, cytokines were measured by flow cytometric analysis in the Bead Assay Flex Sets system. Cytokine levels in pg/mL.*All results are described with standard deviations (SDs).
Here, we show that Citrus/Cydonia comp. has a selective effect on the differentiation of T-cells with regard to the production of cytokines; the production of IL-10 is relatively larger than that of IL-12. By that, Citrus/Cydonia comp. also seems to have an effect on the induction of regulatory (IL-10 producing) T-cell subsets. Hence, as a consequence, Citrus/Cydonia comp. might be producing an allergy reducing effect, at which it does not concern Th1 induction and the reduction of the allergen-specific Th2 response, bearing the risk of induction of a chronic inflammation and very likely even an increased risk for autoimmunity.
Recent
developments mainly concern the field of allergen-specific immunotherapeutic
protocols. This immunotherapy is widely believed to occur through restoration
of the disturbed Th1-Th2 balance [
Citrus/Cydonia comp. is likely to induce more regulatory T-cells, whether CD4+CD25+Fosp3+ natural or antigen-induced IL-10 and/or TGF-b producing Tr-cells, that are, therefore, very immunosuppressive, and which are capable of reducing allergen specifically activated Th2 cells. Our results imply that Citrus/Cydonia comp. does not induce a complete state of immunosuppression, resulting in a diminished resistance against infections and a reduced protection against tumors. This is consistent with the long-term clinical experiences and the results of the empirical studies on the use of Citrus/Cydonia comp.
Based on these in vitro investigations, we hypothesized that Citrus/Cydonia comp. is capable of neutralizing (to some extent) the changes, characteristic to allergic rhinitis, with regard to the construction, the maturation, the differentiation, and the activity of the immune system. By that, it is possible to explain the therapeutic positive effects on allergic rhinitis patients treated with Citrus/Cydonia comp. found in previous studies and clinical practice.
The conclusions based on this study are of great importance, since the standard treatment of allergic rhinitis is based on the long-term use of antihistamines, potentially in combination with a local application of corticosteroids, in case of persisting and/or serious symptoms. Those treatments tend to reduce the symptoms, but they do not possess any immunotherapeutic potency themselves. This implies that it is compulsory for individual patients to keep on using such medicines for many years. Based on our pilot data, indicating that in vitro Citrus/Cydonia comp. is capable of modulating the Th1-Th2 balance, we actually expect Citrus/Cydonia comp. to have an immunotherapeutic potency. This adds to the clinical therapeutic effect from Citrus/Cydonia comp., both as an injection and as a topical application. This implies that a long-term treatment with Citrus/Cydonia comp. injections during several years, before the start of the pollen season, can potentially restore the disturbed immune state of rhinitis patients, which essentially could be sufficient to make the allergic complaints disappear.
The authors would like to thank Weleda Netherlands for providing the research medication.