Published online before print May 31, 2007
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
T cells and monocytes in response to unripe apple polyphenolsVeterinary Molecular Biology, Montana State University, Bozeman, Montana, USA
1 Correspondence: Veterinary Molecular Biology, Montana State University, 960 Technology Blvd., Bozeman, MT 59718, USA. E-mail: uvsmj{at}montana.edu
ABSTRACT
Leukocyte adhesion and migration are mediated partially by CD11b/CD18 (membrane-activated complex-1, CR3). Earlier studies have demonstrated a role for green tea polyphenols in down-regulating CD11b on CD8+ T cells and monocytes. We have shown recently a stimulatory effect of unripe apple polyphenols (APP) on 
T cells. Thus, we compared the effect of APP on bovine T cell and monocyte CD11b expression. Purified bovine monocytes and monocyte-depleted PBLs were cultured with APP. CD11b levels decreased on monocytes in response to APP. In contrast, a T cell subset responded to APP by up-regulating CD11b. The CD11b regulation was not seen on T cells or monocytes treated with APP fractions depleted of tannins. The APP-induced down-regulation of CD11b on monocytes was inhibited by an anti-CD11b mAb, consistent with previous studies showing that polyphenols bind CD11b. As expected, the anti-CD11b mAb had no effect on the APP response in resting T cells, as these cells lacked CD11b. Consistent with the changes in surface CD11b expression, APP-treated T cells showed increased adherence to plastic, whereas monocyte adhesion was reduced. APP also induced cytokine gene expression in T cells. Some polyphenols are thought of as anti-inflammatory agents; however, these data, as well as other ongoing studies, indicate they have a proinflammatory effect on T cells. In vivo, plant polyphenols may enhance T cell migration and function at sites of inflammation, where they could induce rapid, immune-regulatory and innate-like immune responses.
Key Words: CR3 • MAC-1 • tannin
Leukocyte adhesion and subsequent migration through the endothelium to sites of inflammation are mediated partially by CD11b. The CD11b/CD18 complex (membrane-activated complex-1, CR3), one of three ß2-integrins, is expressed primarily on NK cells, neutrophils, and monocytes/macrophages [1
]. However, a small population of CD8+ lymphocytes [2
3
] and T cells [4
5
] can express CD11b. The CD11b/CD18 complex can recognize a wide range of ligands, including fibronectin, collagens, laminins [6
], and plant polyphenols [3
].
Plant polyphenols, such as those found in green tea and unripe apples, have been the topic of much research over the past decade. Catechin and tannins, types of polyphenols, display various beneficial, biological activities. For example, unripe apple polyphenols (APP) exhibit antioxidant activities, inhibition of tumor proliferation [7
], and immune modulatory effects [3
8
]. Plant tannins can stimulate macrophages [8
], and green tea polyphenols have been shown to down-regulate CD11b on CD8+ T cells, monocytes, and granulocytes [3
]. APP also stimulates T cells in vitro (Jeff Holderness, manuscript submitted), and feeding of apple-condensed tannins increases the percentage of T cell intraepithelial lymphocytes (IELs) in mice [9
].
As additional research is conducted, it is becoming apparent that T cells have a strong myeloid link in that they express innate receptors [5
], respond rapidly to a variety of antigens [10
11
], and home to epithelial barriers [11
]. As CD11b plays an important role in leukocyte adhesion and migration to sites of inflammation, CD11b expression on macrophages can be regulated by APP, and apple-condensed tannins increase the level of IELs in vivo, we examined the effect of APP on the regulation and function of CD11b on T cells, as compared with monocytes.
The effect of APP on surface CD11b expression was first examined on bovine T cells and monocytes. Monocyte-depleted PBLs and monocytes purified from PBMCs were cultured with 40 µg/ml APP for 24 h. Although most resting T cells expressed no detectable surface CD11b by flow cytometry, a T cell subset in the mixed lymphocyte population responded to APP by up-regulating CD11b (Fig. 1A
). The CD11b+ population of T cells represented an activated population, as indicated by high levels of IL-2R
(65–97% IL-2R+; data not shown). For T cells, the CD11b mean intensity and the percent CD11b+ cells tracked together (Fig. 1B)
; therefore, percent CD11b+ was used to illustrate the regulation of CD11b on T cells throughout this study. A small fraction of the non- T cells in the lymphocyte population did show a slight increase in surface CD11b expression in response to APP (2.4±0.18-fold increase in percent CD11b+; data not shown). However, the increase seen in T cells was much greater (8.9±0.4-fold increase in percent CD11b+). Unlike bovine T cells, the majority of untreated human T cells (70–80% depending on the donor) was CD11b+. However, upon treatment with APP, the percentage of CD11b+ T cells increased (88–96%; data not shown) as seen with bovine cells. The concentrations of APP, which induce CD11b on T cells (20–40 µg/ml) are likely physiologically relevant, as >10 µg/ml in plasma can be obtained following feeding of condensed tannin preparations to rats [14
], and it is assumed that at the point of absorption across the epithelium, concentrations would be much higher.
 View larger version (38K): [in a new window] |
Figure 1. T cells and monocytes differentially regulate surface CD11b in response to APP. PBLs and monocytes were isolated from bovine (1- to 6-month-old male calves) PBMCs by magnetic bead separation (MACS) using anti-CD14-conjugated beads (Miltenyi Biotech, Auburn, CA, USA) [5]. Dried, nonripe apple peel (Apple Poly, LLC, Morrill, NE, USA) was reconstituted in water and sterile-filtered (0.2 µm). Cells were cultured in X-VIVO 15 (Cambrex, Walkersville, MD, USA), with or without APP for 24 h at 106 cells/ml. Flow cytometry was performed using standard techniques [12
] with directly conjugated GD3.8-PE (anti-bovine TCR mAb) and anti-CD11b-FITC mAb (CC126, Serotec, Raleigh, NC, USA). (A) Viable T cells (GD3.8+) and viable monocytes were gated and analyzed for surface CD11b expression. (B) Cells were treated with 0–40 µg/ml APP and analyzed for surface CD11b expression. (C) APP was depleted of tannins using polyvinylpolypyrrolidone (PVPP; Sigma-Aldrich, St. Louis, MO, USA) depletion [13
]. Cells were treated with equivalent concentrations of APP or tannin-depleted APP prior to flow cytometric analysis of surface CD11b. Data are representative of multiple experiments performed with triplicate treatment groups (error bars represent SD). **, Pvalue < 0.01; ***, P value < 0.001.
|
T cells and monocytes in response to APP was dose-dependent (Fig. 1B)
. Toxicity was seen at concentrations above 40 µg/ml APP, especially in the non- T cell population of lymphocytes and monocytes (data not shown). The viability of APP-treated T cells was unaffected by APP at 24 h, as determined by a lack of increased Annexin V staining and no decrease in cell size, as determined by light scatter (data not shown). Similar to green tea polyphenols, specifically epigallocatechin gallate [15
], APP did induce apoptosis of some monocytes after 24 h of stimulation. However, the regulation of CD11b was seen as early as 30 min after APP stimulation (50±19% decrease in CD11b intensity; data not shown), suggesting that the down-regulation of CD11b on monocytes was not a direct result of apoptosis. The active component of the APP extract was removed by passing the extract over PVPP resin (Fig. 1C)
, which binds tannins, suggesting that the active component in the APP was a tannin. Similarly, activity was removed by passage of APP over an LH-20 column. A concurrent study used HPLC to analyze the active component of APP, which confirmed that it was a tannin (J. Holderness, manuscript submitted).
Green tea polyphenols have been shown to bind to CD11b on CD8+ T cells, which prevented adhesion and migration [3
]. We, therefore, tested whether APP similarly bound to CD11b, thus inducing CD11b down-regulation on monocytes. PBLs and monocytes were pretreated separately with PBS, an anti-CD11b mAb, or an isotype-matched mAb prior to stimulation with APP. Monocytes pretreated with the anti-CD11b mAb did not show the typical down-regulation of CD11b, suggesting that the anti-CD11b mAb blocked APP binding to and signaling through CD11b on monocytes (Fig. 2
). As expected, as a result of the lack of surface CD11b on resting T cells, pretreatment with the anti-CD11b mAb did not affect the up-regulation of CD11b on T cells (Fig. 2)
. A slight enhancement of CD11b expression was seen in resting and APP-stimulated T cells, which were pretreated with the anti-CD11b mAb. This could be a result of cross-linking CD11b and thus, inducing up-regulation of the low level of initial surface CD11b. These data suggest that T cells recognize APP through a CD11b-independent pathway, which leads to activation of the cells, resulting in an actual increase in CD11b expression.
 View larger version (27K): [in a new window] |
Figure 2. Pretreatment with an anti-CD11b mAb prevents APP-induced CD11b down-regulation on monocytes but does not affect T cell regulation of CD11b. Bovine PBLs and monocytes, prepared as in Figure 1
, were incubated with PBS, FITC-conjugated anti-CD11b mAb (5 µg/ml), or isotype-matched mAb (IgG2b) in HBSS at 107 cells/ml at 4°C for 30 min. Additional medium (X-VIVO 15), with or without the addition of APP (40 µg/ml), was added to 106 cells/ml and cultured for 24 h at 37°C. Flow cytometry was performed, and viable T cells and monocytes were analyzed for surface CD11b expression. Data are representative of multiple experiments performed with triplicate treatment groups (error bars represent SD). **, Pvalue < 0.01; ***, Pvalue < 0.001.
|
T cells and monocytes in response to APP was examined. T cells in a mixed lymphocyte population and monocytes were incubated in the presence or absence of APP for 24 h. Cultures were then examined for the proportion of suspension versus plastic-adherent cells. Correlating with the CD11b expression data, a greater number of T cells were adherent after APP stimulation (increased CD11b) than without treatment (Fig. 3A
). In contrast, fewer monocytes adhered to the plastic after APP stimulation (decreased CD11b) than in the resting population (Fig. 3A)
. As expected, adherent cells, T cells and monocytes, had higher levels of surface CD11b expression than the correlating suspension populations (Fig. 3B)
. These data suggested that APP regulation of CD11b on monocytes and T cells was functionally relevant to cell adhesion. However, regulation of additional adhesion molecules could have contributed to the changes in the adhesion phenotypes. Studies are currently being performed to examine the affect of APP on additional adhesion molecules. The effect of APP on CD11b varied between T cells and monocytes, as CD11b was maintained on T cells and was functional for at least 24 h in the presence of APP, whereas CD11b was not maintained on monocytes. This difference could be a result of the lower density of surface CD11b on the activated T cells or possibly, differences in CD11b glycosylation between cell types [16
], either of which could have contributed to a reduced interaction of APP with T cell CD11b.
 View larger version (12K): [in a new window] |
Figure 3. APP differentially alters the adhesion phenotype of T cells and monocytes. Bovine PBLs and monocytes were isolated as described in Figure 1
and cultured at 106 cells/ml, with or without APP for 24 h. Suspension cells were obtained by gently washing the wells with PBS/2% horse serum (HS). Adherent cells were obtained by scraping the wells in PBS/HS. Equal amounts of polystyrene 15 µm beads (Polysciences, Warrington, PA, USA) were added to each FACS tube after staining with GD3.8-PE and anti-CD11b-FITC as in Figure 1
. (A) The relative number of suspension or adherent T cells and monocytes was determined by ratio to beads (30,000 events collected) [5]. (B) Surface CD11b expression on suspension and adherent T cells and monocytes was also examined. Data are representative of multiple experiments performed with triplicate treatment groups (error bars represent SD). *, Pvalue < 0.05; **, P value < 0.01; ***, Pvalue < 0.001.
|
T cells. In our concurrent study (J. Holderness, manuscript submitted) and as indicated here, we have found that IL-2R expression on T cells was up-regulated by APP. Additional evidence of APP-induced activation of T cells was then determined by examining its impact on cytokine gene expression. As shown in Figure 4
, APP treatment increased expression of several cytokine transcripts in purified (>98%) bovine T cells after 4 h of treatment. This cytokine profile was similar to that of T cells treated with other inflammatory agents, such as LPS or peptidoglycan [5
]. It is interesting that APP did not induce expression of TNF- (Fig. 4)
and IFN- transcripts (data not shown). The mechanism of APP signaling on T cells leading to these changes is unknown. As mentioned above, signaling through CD11b does not account for the response by T cells. Current experiments are focused on defining signal transduction pathways, which are triggered in T cells by APP.
 View larger version (13K): [in a new window] |
Figure 4. T cells up-regulate cytokine transcripts in response to APP treatment. Bovine PBMCs were stained with GD3.8-PE and sorted to >98% purity on a VANTAGE SE cell sorter (BD Biosciences, San Jose, CA, USA). Purified T cells were rested overnight at 106 cells/ml in X-VIVO 15 prior to 4 h of stimulation, with or without APP. Cells were harvested and RNA extracted using QiaShredder columns and the RNeasy RNA extraction spin columns as per the manufacturer’s protocol (Qiagen, Valencia, CA, USA). Real-time RT-PCR was performed as described previously [12
] using the ABI PRISM 7500 sequence detection system to detect SYBR Green incorporation (Applied Biosystems, Foster City, CA, USA). Data were normalized to 18S levels and are representative of multiple experiments performed with triplicate treatment groups (error bars represent SD). All comparisons have a Pvalue <0.01. VEGF, Vascular endothelial growth factor; iNOS, inducible NO synthase.
|
T cells are capable of rapid, innate-like responses, as well as having immune-regulatory roles, thus making them integral to immune responses at sites of infection and inflammation. Polyphenol-induced up-regulation of surface CD11b on T cells and its impact on recruitment, combined with our recent finding that polyphenols also up-regulate proliferative responses of these cells (J. Holderness, manuscript submitted), may help explain the increased levels of T cells at the gut epithelium in mice fed apple-condensed tannins [9
]. Additional in vivo studies are being performed to examine the effects of APP on T cell recruitment and activation and whether enhanced homing of T cells to and function at mucosal barriers affects the course of mucosal infections.
ACKNOWLEDGEMENTS
Funding for these studies was provided by National Institutes of Health (NIH), National Institutes of Allergy and Infectious Diseases, #HHSN26620040009C N01-AI-40009, NIH Centers of Biomedical Research Excellence, and the Montana Agriculture Experiment Station. M. A. J. holds shares in LigoCyte Pharmaceuticals, which together with Montana State University, holds a NIH contract that partially funded the work presented in this article. Dr. Mark Quinn and Kirk Lubick are thanked for their constructive comments about this manuscript.
Received February 23, 2007; revised April 23, 2007; accepted May 9, 2007.
REFERENCES
and CD4+ ß T lymphocytes, which respond to Anaplasma marginale, demonstrates higher expression of chemokines and other myeloid cell-associated genes by WC1+ T cells J. Leukoc. Biol. 80,939-952 T cells respond directly to pathogen-associated molecular patterns J. Immunol. 174,6045-6053/ T cells, their T cell receptor usage and role in human diseases Springer Semin. Immunopathol. 21,55-75[Medline] T cells in host defense and epithelial cell biology Clin. Immunol. Immunopathol. 86,121-133[CrossRef][Medline] T lymphocyte subsets defines unique tissue-specific functions J. Leukoc. Biol. 73,306-314
B activation Mutat. Res. 480-481,243-268This article has been cited by other articles:
 |
|
 |
|
  J. Holderness, L. Jackiw, E. Kimmel, H. Kerns, M. Radke, J. F. Hedges, C. Petrie, P. McCurley, P. M. Glee, A. Palecanda, et al. Select Plant Tannins Induce IL-2R{alpha} Up-Regulation and Augment Cell Division in {gamma}{delta} T Cells J. Immunol., November 15, 2007; 179(10): 6468 - 6478. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
