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Journal of Leukocyte Biology logoLink to Journal of Leukocyte Biology
. 2009 Jan 7;85(4):606–616. doi: 10.1189/jlb.0608338

Superantigen-mediated differentiation of bovine monocytes into dendritic cells

Keun Seok Seo *, Joo Youn Park †, William C Davis ‡, Lawrence K Fox †, Mark A McGuire §, Yong Ho Park ‖, Gregory A Bohach *,1
PMCID: PMC2656427  PMID: 19129485

Abstract

Although many effects of staphylococcal superantigens (SAg) on T cells are well established, less is known about their effects on APC. In this study, bovine PBMC were stimulated with a low dose of staphylococcal enterotoxin C1 (SEC1). The phenotype of adherent cells (Ac) derived from bovine PBMC cultured with SEC1 [SEC1-stimulated Ac (sAc)] for 192 h was CD14−, CD68−, CD163−, dendritic cell (DC)-specific ICAM-3-grabbing nonintegrin+, MHC class II (MHC II)high, CD11alow, CD11bhigh, CD11chigh, and CD1bhigh, suggesting these cells were dendritic cells (DC). SEC1 also induced transcription of the CXCL1, -2, and -3 family, CXCL6, CCL2, and CCL5 genes in sAc, which increased rapidly but returned to basal levels by 48 h. In contrast, increased transcription of CCL3, CCL8, and CXCL12, responsible for mononuclear cell migration and chronic inflammation, was sustained. In vitro cell migration assays showed vigorous migration of granulocytes, followed by migration of mononuclear cells. The autologous MLR showed that sAc induced a dose-dependent proliferation of CD4+ T cells and an even stronger proliferation of CD8+ T cells. This effect was inhibited or reduced by pretreatment with mAb to CD11b, MHC II, or MHC II plus CD18. These results indicate that stimulation of bovine PBMC by SAg induces differentiation of monocytes into DC.

Keywords: staphylococcal enterotoxin C1, chemokines, leukocyte trafficking, autologous MLR

INTRODUCTION

Staphylococcus aureus is a broad host-range pathogen that causes diseases ranging from toxic shock to mild skin infections in humans and mastitis in dairy cows [1]. S. aureus produces a variety of virulence factors including cytotoxins, hemolysins, nucleases, lipases, hyaluronidase, collagenase, and staphylococcal enterotoxins (SE) [1]. Recent studies have shown that most S. aureus isolates from bovine mastitis produce one or more SE, suggesting that SE have a role in pathogenesis of this infection [2]. SE are a prototypic group of microbial superantigens (SAg). Numerous studies have demonstrated that SAg induce extensive proliferation of T cells, typically mediated by cross-linking of the variable region of the β chain of the TCR with MHC class II (MHC II) molecules [3, 4]. Costimulatory signals induced by interaction of CD28/B7 (CD80), CD18/ICAM-1 (CD54), and CD2/CD58 are required for maximal T cell proliferation [5,6,7]. Recently, we demonstrated that stimulation of bovine PBMC with a relatively low dose of SEC1 (5 ng/ml) for 240 h induces proliferation of CD4+ and CD8+ T cells. The CD4+ CD25+ T cell subset that developed following SEC1 stimulation exhibited immunosuppressive activity. Add-back experiments demonstrated that these cells could suppress the response of naïve T cells to a heat-killed, fixed S. aureus antigen. We concluded from these studies that the cells were likely bovine regulatory T cells (Tr) [8].

APC provide critical signals in SAg-induced T cell proliferation. Despite their importance, limited information has been obtained regarding SAg-mediated changes in the phenotype and function of APC. Studies in humans and other animals have demonstrated that following exposure to SAg, APC produce high levels of proinflammatory cytokines and chemokines such as IL-1α, IL-1β, TNF-α, IL-8, IL-10, IL-12, CCL3, and CCL5 [9,10,11,12]. In mice, SEA and SEB induce acute migration of neutrophils to the site of injection. Expression of TNF-α and ICAM-1 preceded expression of chemokines [13]. Injection of SEB in mice induces the maturation of dendritic cells (DC), which up-regulate MHC II, CD40, CD80, CD86, and CD205 expression [14].

Although the effect of SAg on bovine APC has not been well-documented, several approaches have been used to elucidate the proinflammatory effect of SAg in bovine mastitis. Soltys and Quinn [15] demonstrated that selective migration of CD4+ T cells, γδ+ T cell subsets, and neutrophils to the mammary gland occurs during staphylococcal mastitis. However, the recruitment mechanism and functional characteristics of these cells were not determined. Kuroishi et al. [16] demonstrated that intramammary infusion of SEC1 induces a rapid migration of neutrophils into milk within 24 h. They also demonstrated that in vitro stimulation of bovine PBMC with SEC1 increased transcription of IL-8 and CXCL3 significantly.

The finding that SEC1 induces a strong, acute, proinflammatory, and chronic immunomodulatory response led us to postulate that unique phenotypic and functional changes also occur in bovine APC. As shown in the present study, exposure to SEC1 induces differentiation and functional alterations in APC, resulting in a phenotype characteristic of DC. These bovine DC exhibit properties that could influence the outcome of exposure to a SAg-expressing strain of S. aureus.

MATERIALS AND METHODS

Animals and reagents

Blood was obtained from purebred adult, healthy Holstein-Frisian steers (18–24 months old) via jugular vein venipuncture. Animals were maintained according to the Association for the Assessment and Accreditation of Laboratory Animal Care and regulations established by the Animal Care and Animal Use Committee at the University of Idaho (Moscow, ID, USA). SEC1 was purified from cultures of S. aureus RN4220 (pMIN121) as described previously [17]. mAb used in phenotypic characterization by flow cytometry (FC), microscopic analysis, and blocking assays are listed in Supplemental Table 1. Note that anti-human CD68 mAb, anti-pig CD163 mAb, and polyclonal antibody to human DC-specific ICAM-grabbing nonintegrin (SIGN) cross-react with the bovine ortholog of CD68, CD163, and DC-SIGN, respectively [18,19,20].

Cell stimulation

Total leukocytes or PBMC were obtained by density gradient centrifugation using Ficoll-Hypaque (Amersham Pharmacia Biotech, Uppsala, Sweden) according to standard techniques [17]. PBMC were adjusted to a concentration of 1 × 106 cells/ml in complete culture medium [CCM; RPMI-1640 medium (Invitrogen, Gaithersburg, MD, USA), supplemented with 10% heat-inactivated FBS (HyClone, Logan, UT, USA), 2 μM L-glutamine (Invitrogen), 100 U/ml penicillin/streptomycin (Invitrogen), and 50 μM 2-ME (Sigma Chemical Co., St. Louis, MO, USA)] and cultured in tissue-culture polystyrene Petri dishes (Falcon, Lincoln Park, NJ, USA) for 6 h. SEC1 (5 ng/ml final concentration) was added, and the cultures were maintained up to 240 h. At designated time-points (3, 6, 9, 24, 48, 96, 144, 192, or 240 h) non-adherent cells (non-Ac) were removed by washing three times with CCM. The remaining SEC1- stimulated Ac (sAc) were harvested, and culture supernatants, removed prior to washing, were frozen for use in other assays. Naive Ac (nAc) were harvested as described above but from cultures not exposed to SEC1.

FC analysis

nAc and sAc were analyzed by indirect immunofluorescence staining and FC. Briefly, nAc and sAc were incubated with the appropriate mAb at 4°C for 30 min, followed by incubation with isotype-specific, anti-mouse antibodies conjugated to FITC or PE (Invitrogen) at 4°C for 30 min in the dark. After washing, cells were analyzed with a FACSAria flow cytometer equipped with FACSDiva (BD Biosciences, Bedford, MA, USA) and Flowjo software (Tree Star, Ashland, OR, USA).

Microscopic analysis

For the phase-contrast microscopy assay, PBMC were cultured in tissue- culture polystyrene Petri dishes in the presence or absence of SEC1 as described above. After removal of non-Ac, live images of nAc and sAc were obtained using a Nikon Eclipse TS100 inverted phase contrast microscope while viewing at 200×, 400×, or 600× magnification (Nikon, Melville, NY, USA). In some experiments, nAc and sAc were fixed with methanol and stained with Wright-Giemsa solution (Sigma Chemical Co.). For fluorescence microscopy, PBMC (1×106) were cultured in Lab-Tek® chambered cover-glass slides (borosilicate cover-glass, Nunc, Rochester, NY, USA) in the presence or absence of SEC1. After thorough washing to remove non-Ac, nAc and sAc were labeled with an anti-CD14 or an anti-DC-SIGN mAb, followed by labeling with an isotype-specific anti-mouse antibody conjugated to FITC or anti-goat antibody conjugated to Alexa Fluor 594 (Invitrogen). The cells were then fixed in 2% PBS-buffered formaldehyde. Fluorescence images were obtained with a Zeiss LSM5 Pascal confocal-scanning microscope equipped with a Plan-Apochromat 63×/1.4 Oil differentiated interface contrast (DIC) lens and argon (Ar) and helium-neon (HeNe) lasers at 630× or 1260× magnification (Zeiss, Thornwood, NY, USA).

Quantitative real-time-PCR (QRT-PCR)

RNA was extracted from nAc or sAc (∼5×106) using Trizol reagent (Invitrogen). First-strand cDNA was generated from 1 μg RNA using Superscript II RT (Invitrogen) and oligo dT primers (Invitrogen). The RT reaction was performed in a 20-μl vol, according to the manufacturer’s specifications. Primers for QRT-PCR were designed with Primer Express Version 2.0 (Applied Biosystems, Foster City, CA, USA) based on GenBank sequences (Supplemental Table 2). The QRT-PCR reactions were conducted using the SYBR Green I dye master mix and an ABI Prism 7500 Real-Time PCR System (Applied Biosystems). QRT-PCR data were analyzed using Sequence Detector Systems software, Version 1.2.2 (Applied Biosystems). The threshold cycle (CT) was determined by the Sequence Detector Systems software. CT were then normalized by calculating ΔCT [CT of target-CT of the internal control (β- actin gene)]. Normalized ΔCT data were used in the comparative CT method (ΔΔCT) of calculating ΔΔCT = [ΔCT of designated time- point–ΔCT of 0 h], according to the manufacturer’s instructions.

In vitro cell migration assay

The chemotactic activity of supernatants from PBMC (or media controls), cultured with SEC1 for various lengths of time, were measured using 24-well culture inserts (pore size, 3 μm, BD Biosciences). Cell culture inserts containing 1 × 106 total leukocytes in RPMI medium were added to individual wells. The cell preparations were cultured at 37°C for 30 min. Cells migrating through the membrane were harvested and counted using a hemacytometer after staining with Trypan blue. The distribution of various subpopulations, among the cells that migrated through the membrane, was analyzed by FC using mAb specific for CD2, CD14, or granulocytes.

Pinocytosis assay

nAc or sAc (192 h; 1×105) were obtained from PBMC cultures and cocultured with FITC-Dextran particles of various sizes (40, 70, and 500 kDa, Molecular Probes, Eugene, OR, USA) at 37°C for 30 min. After washing with PBS, FITC-Dextran uptake was analyzed by FC.

Autologous MLR

The autologous MLR is a useful in vitro tool for the investigation of T cell proliferation in response to stimulation with autologous non-T cells, for example, DC. To assess the T cell proliferation-inducing ability of Ac, autologous MLRs were performed. nAc or sAc at 192 h were harvested and washed three times (see above). The washed nAc were then labeled with mAb specific for CD2, B lymphocytes, or the TCR δ chain, followed by PE-conjugated, isotype- specific anti-mouse antibodies. The washed sAc were also labeled with mAb listed above, plus a CD14 mAb. After washing, the cells were incubated with anti-PE- labeled micro-beads (Miltenyi Biotec, Auburn, CA, USA) for 15 min at 4°C. Labeled cells were sorted by negative selection using a LS column (Miltenyi Biotec), according to the manufacturer’s instructions. In some experiments, sorted sAc, harvested after 192 h, were treated with 2% PBS-buffered formaldehyde for 10 min to generate fixed sAc (fsAc). To enrich the naïve T cell (nTc) population, PBMC were allowed to adhere to polystyrene Petri dishes. Non-Ac were harvested after 6 h incubation in CCM. Cells were then labeled with mAb specific for CD14, B lymphocytes, or CD45R0. Labeled cells were sorted by negative selection as described above. As assessed by FC, CD14+, B lymphocytes, or CD45R0+ cells were not detected among nTc in three separate experiments (data not shown). Various numbers (1×103, 5×103, 1×104) of sorted nAc, sAc, or fsAc were seeded into the 24-well culture plates (Corning Life Sciences, Nagog Park Acton, MA, USA) containing nTc (1×105) and then cultured for 96 h. Proliferation of nTc was assessed by incorporation of the [3H]- thymidine assay. Briefly, [3H]-thymidine (1 μCi/well) was added after 72 h of culture. Cells were harvested 18 h later. Incorporation of [3H]-thymidine into cellular DNA was measured by liquid scintillation counting [17].

For phenotypic analysis of cocultured nTc, these cells (1×105) were cocultured with sAc (1×104). Cocultures were maintained for 96 h and then labeled with mAb specific for T cell surface markers [CD4, CD8, γδ TCR, CD25, CD26, CD45R, and CD62 ligand (CD62L)] as described above. In some experiments, nTc (1×105) were incubated with CFSE (Molecular Probes) prior to coculture with sAc as described previously [8].

To help assess whether proliferation of nTc induced by sAc could be attributable to the presence of residual SEC1 in sAc suspension, nTc (1×105) were cocultured with sAc (1×104) or nAc (1×104) plus SEC1 (5 ng/ml) in the absence or presence of the mAb cocktails (anti-MHC II DR, DQ, and/or CD18 or CD11a, CD11b, or CD11c) or isotype control mAb at a 1 μg or 5 μg/ml final concentration. Cultures were maintained for 96 h. Proliferation of nTc was assessed by incorporation of [3H]-thymidine as described above.

Statistical analysis

Data acquired in autologous MLR experiments were analyzed with the ANOVA test by calculating the differences of least square means (LSM) using SAS software (Version 9.1.2 for Windows, SAS Institute Inc., Cary, NC, USA).

RESULTS

Bovine monocytes differentiate into DC following stimulation with SEC1

FC was used to monitor the relevant phenotypes of SEC1-sAc. Prior to stimulation, the majority of nAc expressed CD14 and CD163 (>95%) or CD68 (56.1%), suggesting that they were monocytes or macrophages (Fig. 1 and Supplemental Table 3). CD11a, CD11b, and MHC II were also expressed by these cells; however, they did not express CD1b (Fig. 2A). Interestingly, CD11c, a phenotypic marker typical of DC in humans and other well-studied animals, was expressed by these cells. The activation markers CD40, CD86, and CD172a were expressed. CD25, CD69, and CD80 were not detected (Fig. 2B).

Fig. 1.

Fig. 1.

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Analysis of markers specific for monocytes, macrophages, and DC. Before or after bovine PBMC were stimulated with SEC1 (5 ng/ml) for 240 h, nAc or sAc were harvested and analyzed by FC using mAb specific for monocytes (CD14 and CD163), macrophages (CD68), or DC (DC-SIGN). In the 0 h row, dotted lines represent isotype controls, and solid lines indicate the results using mAb specific for the surface markers. In the other panels, dotted lines represent the data obtained at 0 h, and the solid lines represent the results obtained at the indicated time-points. The data shown are from a single representative experiment that was conducted three times.

Fig. 2.

Fig. 2.

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Phenotypic characteristics of sAc. Before or after bovine PBMC were stimulated with SEC1 (5 ng/ml) for 240 h, nAc or sAc were harvested and analyzed by FC using mAb specific for several phenotypic (A) or activation markers (B). In the 0 h rows, dotted lines represent isotype controls, and solid lines indicate the results using mAb specific for the surface markers. In the other panels, dotted lines represent the data obtained at 0 h, and the solid lines represent the results obtained at the indicated time-points. The data shown are from a single representative experiment that was conducted three times.

Following exposure to SEC1 for 240 h, most sAc became negative for CD14, CD68, and CD163 (>90.7% at 240 h), whereas expression of DC-SIGN increased gradually (89.4%±2.3 at 240 h; Fig. 1 and Supplemental Table 3). Expression of CD11a increased until 96 h and then, decreased dramatically (Fig. 2A). Expression of CD11b and CD11c increased gradually until 144 h and then slightly decreased (Fig. 2A). After 192 h, most sAc were CD14−, CD68−, CD163−, DC-SIGN+, MHC IIhigh, CD11alow, CD11bhigh, CD11chigh, and CD1bhigh. Although CD11c and CD1b are not DC-specific markers, the overall phenotype observed at 192 h was similar to DC in humans and other animals, suggesting that SEC1 induced differentiation of monocytes into DC.

Following exposure to SEC1, CD25 and CD69 levels in sAc increased gradually; CD40 and CD80 increased gradually until 144 h and then decreased (Fig. 2B). The expression of CD172a increased slightly until 96 h and then decreased dramatically (Fig. 2B). However, CD86 expression was not altered significantly throughout the culture period (Fig. 2B). These observations suggested that SEC1 stimulation not only induced differentiation of monocytes into DC but also induced maturation of DC.

sAc develop morphological characteristics typical of DC

For phase-contrast microscopic analysis, bovine PBMC were cultured on polystyrene plastic cell culture Petri dishes and viewed with an inverted microscope. Prior to SEC1 stimulation, freshly prepared nAc appeared small and round in shape (Fig. 3, A and C). Following stimulation with SEC1 for 192 h, sAc increased in size and developed long, fine dendrites (Fig. 3, B and D). Higher magnification (600×) revealed that the enlarged cells characteristically contained an eccentric nucleus and an increased amount of cytoplasm (Supplemental Fig. 1A).

Fig. 3.

Fig. 3.

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Microscopic analysis of sAc. Images were obtained before (nAc-0 h column) and after bovine PBMC were stimulated with SEC1 for 192 h (sAc-192 h column) in tissue-culture polystyrene Petri dishes (A–D) or in Lab-Tek® chambered cover-glass slides (E–L). Images shown are of nAc and sAc (A and B) or Giemsa-Wright-stained cells from the same cultures (C and D) observed by inverted light microscopy (A and C at 400× original magnification; B and D at 200× original magnification), confocal microscopy using the phase mode (E and F), the fluorescence mode to assess expression of CD14 (G and H), the fluorescence mode to assess expression of DC- SIGN (I and J), or combined images (K and L) obtained at 630× original magnification. Results shown are from a single representative experiment that was conducted three times.

For confocal microscopy, bovine PBMC were cultured on borosilicate cover- glass slides. Prior to SEC1 stimulation, freshly prepared nAc had irregular shapes with short, cytoplasmic processes, expressed high levels of CD14, and were negative for DC-SIGN (Fig. 3, E, G, and K). Following exposure to SEC1 for 192 h, expression of CD14 was diminished or absent, whereas that of DC-SIGN was increased highly (Fig. 3, J and L). Under higher magnification (1260×), sAc showed numerous fine dendrites on the cell surface (Supplemental Fig. 1B).

SEC1 induces proinflammatory cytokines and a distinct chemokine response

Transcription of key proinflammatory cytokine and chemokine genes of nAc and sAc was analyzed by QRT-PCR. Transcription of IL-1α, IL-1β, and IL-6 increased within 3 h after stimulation (2.7- to 8.1-fold), peaked at 6–9 h, and then decreased gradually. This effect was more dramatic for IL-1β after 48 h (decreased ∼776-fold; Fig. 4A). Transcription of TNF- α and GM-CSF also increased and persisted (2.7- to 12.8-fold; Fig. 4, A and B). Transcription of IL-12 and IL-18 increased similarly until 48 h; transcription of IL-12 continued to increase (∼113.7-fold at 96 h), and that of IL-18 decreased gradually (Fig. 4B). Transcription of IL-10, an anti- inflammatory cytokine, remained slightly below the basal level (Fig. 4B).

Fig. 4.

Fig. 4.

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Transcription of proinflammatory and immunomodulatory cytokines and chemokines in sAc. Bovine PBMC were stimulated with SEC1 (5 ng/ml) for 240 h. Transcriptional change of proinflammatory and immunomodulatory cytokines and chemokine genes in nAc or sAc was measured by QRT-PCR. Relative transcription of these genes in sAc was assessed by the ΔΔCT method (see Materials and Methods). QRT-PCR results shown represent the means ± sem of data combined from three separate experiments (n=9).

Transcription of chemokines (the CXCL1, -2, and -3 family, CXCL6, and IL-8) associated with migration of granulocytes, particularly neutrophils, increased rapidly and peaked at 9 h (1.34- to 2.5-fold) and then declined to below the basal level by 48 h (Fig. 4C). Transcription of chemokines (CXCL12, CCL3, and CCL8) also increased early and remained elevated (2.9- to 22.9-fold increase) during the entire period of culture (Fig. 4D).

Chemokines induced by SEC1 induce differential leukocyte migration

The finding that SEC1 induces distinct chemokine responses, before and after 48 h of culture, suggested that the toxin might temporally induce dependent differences in leukocyte migration. Consistent with chemokine transcription results, cell migration assays demonstrated a strong induction of granulocyte migration by culture supernatants harvested up to 24–48 h poststimulation (Fig. 5). A gradual shift toward an increase in CD14+ and CD2+ cell migration and a corresponding decrease in granulocyte migration occurred with culture supernatants obtained from later time-points (at 96–240 h). The total number of cells that migrated through the membrane and the frequency of CD2, CD14, and granulocytes in the preparations are presented in Supplemental Table 4.

Fig. 5.

Fig. 5.

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Differential leukocyte migration induced by SEC1-stimulated PBMC culture supernatants. Leukocyte migration in vitro was assayed using 3 μm pore cell culture inserts. Culture supernatants harvested at the designated times were added to wells and leukocytes (in RPMI medium) were added to the cell culture inserts. Cells migrating out of the insert were quantified and characterized by FC analysis using mAb specific for CD2, CD14, and granulocytes. Dotted lines represent isotype control, and solid lines indicate the cells reacting with each mAb. No significant cell migration was observed with RPMI medium control (data not shown). The data shown are from a single representative experiment that was conducted three times.

Pinocytosis by sAc is significantly higher than that by nAc

Pinocytosis of FITC-dextran was measured as an indication of potential antigen uptake ability. Following coincubation with the FITC-dextran, the fluorescence intensity of sAc was significantly higher (1.2–2.3 log10-fold) than that of nAc (P<0.01; Fig. 6).

Fig. 6.

Fig. 6.

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Pinocytosis of nAc or sAc assessed by FITC- Dextran uptake. nAc or sAc harvested after 192 h were cocultured with FITC- Dextran of various sizes (40, 70, or 500 kDa) at 37°C for 30 min. The dotted lines represent nAc without FITC-Dextran (control). The shadowed areas indicate the results obtained with nAc containing FITC-Dextran. The unshadowed areas enclosed by solid lines indicate the results obtained with sAc, containing FITC- Dextran. Results shown are from a single representative experiment that was conducted three times.

sAc elicit a strong, autologous CD8+ T cell proliferation response

One of the properties of DC is the ability to activate nTc. To test whether sAc induce proliferation of nTc, the autologous MLR was performed by coculturing nAc or sAc with nTc, which proliferated in the presence of sAc in a dose-dependent manner but in the absence of antigen (Fig. 7; sAc+nTc panel). The proliferation of nTc promoted by sAc peaked at a ratio of 1:10 and was significantly higher than the response observed with nAc in the presence of 5 ng/ml SEC1. In contrast, neither nAc nor fsAc induced proliferation of nTc (Fig. 7; nAc+nTc and fsAc+nTc panels).

Fig. 7.

Fig. 7.

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Autologous T cell proliferation by sAc. Before or after bovine PBMC were stimulated with SEC1 (5 ng/ml) for 192 h, nAc or sAc were harvested and sorted using magnetic beads. In the autologous MLR, various numbers (1×103, 5×103, or 1×104) of nAc, sAc (harvested at 192 h), or fsAc (harvested at 192 h) were cocultured with nTc (1×105), depleted of memory T cells expressing CD45RO in the presence or absence of SEC1 (5 ng/ml). After 96 h, nTc proliferation was assessed by measuring [3H]-thymidine incorporation. The results shown are the mean of triplicate measurements ± sem of data combined from three separate experiments (n=9). Shared letters represent datasets in which the LSM are not statistically significant (P<0.001).

FC analysis showed that the proportion of CD8+ T cells increased dramatically from 18.0 ± 2.3% to 53.5 ± 4.8% (Fig. 8A). This caused an inversion of the CD4:CD8 ratio from a normal ratio of 1.79 ± 0.64 before the coculture to 0.45 ± 0.28 after 96 h (Fig. 8A). In contrast, the proportion of CD4+ T cells decreased from 32.3 ± 3.7% to 23.9 ± 2.4%, and γδ+ T cells were reduced from 34.3 ± 4.2% to 10.9 ± 2.7% after 96 h in the autologous MLR (Fig. 8A).

Fig. 8.

Fig. 8.

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sAc promote CD8+ T cell proliferation in the autologous MLR. (A) nTc and sAc (harvested at 192 h) were cocultured for 96 h. Major T cell subpopulations in cocultures were analyzed by FC. In the 0 h row, dotted lines represent isotype controls, and solid lines represent the results using mAb specific for the indicated T cell markers. In the 96 h row, dotted lines represent the cell-labeling data at 0 h, and solid lines represent the results at 96 h. (B) Prior to coculture with sAc, nTc were labeled with CFSE and then cocultured for 96 h. Dotted lines represent the cell- labeling data at 0 h, and solid lines represent the results at 96 h. Numbers in the upper-right corners and those in the upper-left corners represent the percentage of undivided and divided cells, respectively. Results shown are from a single representative experiment that was conducted three times.

To determine if the increased proportion of CD8+ T cells in the autologous MLR was attributed to their enhanced proliferation, nTc were labeled with CFSE prior to the autologous MLR. As shown in Figure 8B, CD8+ T cells proliferated to a greater extent than CD4+ T cells (90.6% and 69.9%, respectively), as indicated by a reduction in CFSE staining. This indicated that the reversal of the CD4:CD8 ratio was a result of a comparatively more extensive proliferation of CD8+ T cells rather than a reduction in the number of CD4+ and γδ+ T cells.

The phenotypes of proliferating CD4+ and CD8+ T cells in the autologous MLR were analyzed by FC (Fig. 9). Expression of CD25 and CD26 (T cell activation markers) increased, and that of CD62L and CD45R (naïve T cell markers) decreased in both populations, indicating that CD4+ and CD8+ T cells were activated in the autologous MLR.

Fig. 9.

Fig. 9.

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Phenotypic characteristics of (A) CD4+ and (B) CD8+ T cells induced by sAc. In the 0 h rows, dotted lines represent the isotype control, and solid lines represent results using mAb specific for T cell activation markers (CD25 and CD26) or naïve T cell markers (CD62L and CD45R). In the 96 h rows, dotted lines represent the data for surface markers at 0 h, and the solid lines represent the T cell marker-labeling results at 96 h. The results shown are from a single representative experiment that was conducted three times.

Role of MHC II and CD11/CD18 integrins in the autologous MLR

mAb specific for MHC II and CD11/CD18 integrins were used to determine the role of these molecules in activation and proliferation of nTc induced by sAc or SEC1. As shown in Figure 10A, T cell proliferation by sAc was essentially blocked completely by the anti-CD11b mAb (at concentrations of 1 or 5 μg/ml), the anti-MHC II mAb (at 5 μg/ml), or a combination of anti-MHC II and CD18 mAb (5 μg/ml each). The anti-CD11a mAb (5 μg/ml) alone also inhibited proliferation to levels of 18.6% compared with the isotype control (Fig. 10A). However, anti-CD18 or CD11c mAb (5 μg/ml) only inhibited proliferation to levels of 44.1% and 53.1%, respectively, compared with the isotype control (Fig. 10A).

Fig. 10.

Fig. 10.

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Mechanisms of sAc-induced T cell proliferation differ from that of SEC1. (A) sAc at 192 h (1×104) were cocultured with nTc (1×105) in the absence or presence of mAb specific to MHC II DR, DQ, and/or CD18 or CD11a, CD11b, or CD11c or irrelevant isotype control antibody (1 μg or 5 μg/ml). (B) nAc (1×105) were cocultured with nTc (1×105) plus SEC1 (5 ng/ml) in the absence or presence of mAb described above. After 96 h, the proliferation of nTc was assessed by measuring [3H]-thymidine incorporation. The results shown are the means of triplicate measurements ± sem of data combined from three independent experiments (n=9). Shared letters represent datasets in which the LSM are not statistically significant (P<0.001).

In contrast to proliferation induced by sAc, SEC1-induced proliferation of nTc was inhibited completely in the presence of the anti-CD11a or anti-CD18 mAb (at 5 μg/ml; Fig. 10B). Also, in contrast to proliferation induced by sAc, the anti-CD11b mAb resulted in only partial inhibition to levels of ∼77.0% at 1 μg/ml and 48.2% at 5 μg/ml compared with the isotype control (Fig. 10B). The proliferation response was not affected by adding isotype control mAb in either assay.

DISCUSSION

Several lines of evidence suggest that SAg play a role in chronic staphylococcal mastitis [8, 13, 21,22,23]. The SEC1 dose and times of exposure used in this study were established to mimic the conditions in chronic mastitis. For example, it was shown that experimental intramammary infection by SAg-producing S. aureus isolates results in chronic infections lasting more than 2 weeks and SAg in the milk at the average concentrations of 2.5–5 ng/ml [16, 24]. We demonstrated previously that exposure of bovine PBMC to a low dose of SEC1 (5 ng/ml) for 144 h induces the development of CD4+ CD25+ Tr and CD8+ CD28− suppressor T cells [8, 23]. These results suggest that long-term exposure to a low concentration of SAg may more closely mimic chronic mastitis associated with SAg-producing S. aureus and is sufficient to induce proinflammatory and immunoregulatory responses. Thus far, efforts to elucidate the mechanisms of pathogenesis induced by SAg have focused primarily on their interaction with T cells. The goal of this current study was to extend our understanding of the effect of SAg on APC and the potential role of these cells in immunomodulation.

This study demonstrated that long-term exposure of bovine PBMC cultures to a representative SAg, SEC1, induces the development of cells with the morphology and phenotypic characteristics of DC, described in humans and other species [25,26,27]. The expression of DC-SIGN and loss of CD14, CD68, and CD163 shown by confocal microscopy and FC in this study strongly suggest that sAc were DC. Some of the observed differences in morphology of nAc and sAc were dependent on the supporting culture matrix. When cells were cultured on polystyrene plastic culture Petri dishes, nAc were round without cytoplasmic processes. Following stimulation with SEC1, the sAc developed long and fine but less numerous cytoplasmic dendrite-like processes. In contrast, when grown on borosilicate cover-glass slides, nAc developed short and thick cytoplasmic processes, and sAc developed numerous small, fine cytoplasmic, dendrite-like processes. Although the culture matrix used affects the morphology of differentiating monocytes, the change in the phenotype of the differentiated monocytes was consistent with differentiation into DC.

The mechanisms by which nAc differentiated into DC in this study were partially revealed. In other species, GM-CSF and IFN-γ or GM-CSF and IL-4 induce differentiation of peripheral monocytes into DC with characteristics of myeloid CD11c+ DC1 or DC2, respectively [25, 28,29,30]. Previously, we demonstrated that bovine PBMC exposed to a low dose of SEC1 induced strong transcriptional up-regulation of GM-CSF and IFN-γ [8], suggesting that these mediators may be involved in SEC1- mediated bovine DC development. Two subsets of bovine afferent lymph DC have been defined based on the differential expression of CD172a [31]. The CD172a+ and CD172a− DC subsets appear to be analogous to DC2 and DC1 in humans, respectively. This prediction is based on the expression of IL-12, which is significantly higher for CD172a+ than CD172a− DC. In the present study, nAc in bovine PBMC uniformly expressed CD172a. Expression of CD172a increased gradually following exposure to SEC1 for up to 96 h and then decreased dramatically after 144 h. This expression pattern was accompanied by the transcription of IL-12, which increased eight- to 16-fold until up to 48 h of culture and then increased dramatically to ∼128-fold during the rest of the culture period. Taken together, the data suggest that a subpopulation of sAc developed into DC with the phenotypic characteristics of bovine CD172a− DC and human DC1 cells.

Cytokines and chemokines play important roles in the acute inflammatory response and differential trafficking of leukocytes. Previous studies in other animal systems revealed that SAg induce a strong, acute, proinflammatory response, characterized by induction of TNF-α, IL-1β, IL-8, CXCL1, CXCL2, CXCL3, and CCL3 [13, 16, 24]. This study revealed that transcription of bovine proinflammatory cytokines and chemokines was up-regulated substantially, following stimulation with SEC1, for up to 48 h. Transcription of chemokines associated with mononuclear cell migration (CXCL12, CCL3, and CCL8) was sustained, and transcription of those associated with migration of granulocytes decreased gradually upon continued exposure of PBMC cultures to SEC1. CXCL12 is a strong lymphocyte and monocyte chemoattractant produced by activated monocytes [32, 33]. CCL3 is a monocyte and T lymphocyte chemoattractant [34], and CCL8 is a monocyte chemoattractant [35]. A common feature of these chemokines is induction of differential migration of mononuclear cell subsets and association with chronic inflammatory disease [36,37,38,39]. Consistent with our findings, studies in humans and rodents demonstrated that activated DC express chemokines related to mononuclear cell migration such as CCL2, CCL3, CCL4, CCL5, and CCL8 and down-regulate the receptors for inflammatory chemokines such as CCR1 and CCR6 [40,41,42].

The transcriptional change in chemokines by SEC1 stimulation was consistent with the in vitro cell migration patterns observed in this study, showing an initial migration of granulocytes followed by migration of mononuclear cells. SEC1 infusion into the bovine intramammary gland caused a rapid increase of granulocytes in milk within 24 h. Later, granulocytes decreased gradually, and CD14+ monocyte and CD8+ T cells in milk increased gradually (manuscript in preparation). This strongly suggests that long-term stimulation by SEC1 eventually induces preferential migration of mononuclear cells, possibly mediated by CCL3, CCL8, and CXCL12. It is noteworthy that the transcription of TNF-α, which mediates production of chemokines by macrophages and expression of adhesion molecules on the endothelial cells [10, 43], increased throughout the culture period in this study. However, transcription of other proinflammatory cytokines greatly increased early but decreased or returned to control levels of transcription at later times during the culture period. This suggests that TNF-α is the secondary mediator for the expression of CXCL12, CCL3, and CCL8 at later time-points.

One of the hallmarks of DC is their ability to induce proliferation of autologous nTc [44,45,46]. This current study demonstrated that sAc have this ability and induce proliferation of nTc in the absence of antigen. Consistent with the prior observation that DC can induce proliferation of CD4+ T cells via signaling through the MHC II and costimulatory molecules [46], we demonstrated that sAc induced vigorous T cell proliferation through MHC II or CD11b. However, we also observed that sAc induced a more vigorous proliferation of CD8+ T cells compared with CD4+ T cells. This could, in part, explain the reversal in CD4:CD8 ratios in SEC1-stimulated PBMC cultures [8].

Several lines of evidence in this study suggest that CD8+ T cell proliferation observed in an autologous MLR assay was not the result of residual SEC1 bound to sAc. First, immunoblot analysis of sAc using hyperimmune, SEC1- specific antiserum (minimal detection limit of 1 ng/ml) indicated that SEC1 was absent or below detectable limits (data not shown). Second, the inversion of the CD4:CD8 T cell ratio occurs after only 96 h of coculture, which is much more rapid than the effect caused by SEC1, which occurred after 240 h, as we reported previously [8]. Third, despite the fact that SAg, present on paraformaldehyde-fixed APC, can induce T cell proliferation [47], fsAc in this study did not induce proliferation of nTc. Fourth, the molecular mechanisms by which sAc induce proliferation of nTc differ from those of SEC1. As shown in Figure 10, A and B, proliferation of nTc induced by sAc was inhibited completely by blocking MHC II or CD11b but not affected greatly by blocking CD18. In contrast, proliferation of nTc induced by SEC1 was inhibited significantly by blocking MHC II, CD18, or CD11a mAb.

Bovine mastitis caused by S. aureus is associated with an initial infiltration of neutrophils, followed by mononuclear cell migration [15]. Results in this study demonstrated for the first time that stimulation by SAg induces differentiation of ruminant monocytes into DC. The functional characteristics of DC induced by SEC1 suggest that DC could contribute to pathogenesis of staphylococcal mastitis by inducing temporally dependent chemokine responses that promote acute and chronic inflammation through differential leukocyte migration into the mammary gland and proliferation of CD8+ T cells with a concomitant CD4:CD8 T cell ratio reversal. These findings, plus the prior demonstration of SAg-induced Tr upon long-term exposure, provide insights into how SAg could influence the outcome of colonization by SAg-producing S. aureus in ruminants and other species.

Supplementary Material

[Supplemental Figure and Tables]
jlb.0608338_index.html (568B, html)

Acknowledgments

This work was supported by the National Institutes of Health grants P20 RR15587, P20 RR016454, and U54AI57141 and grants from the U.S. Department of Agriculture National Research Initiative and the Idaho Agricultural Experimental Station. We thank A. Norton (Department of Microbiology, Molecular Biology and Biochemistry, University of Idaho) for technical help with confocal microscopy.

References

  1. Novick R P. Autoinduction and signal transduction in the regulation of staphylococcal virulence. Mol Microbiol. 2003;48:1429–1449. doi: 10.1046/j.1365-2958.2003.03526.x. [DOI] [PubMed] [Google Scholar]
  2. Smyth D S, Hartigan P J, Meaney W J, Fitzgerald J R, Deobald C F, Bohach G A, Smyth C J. Superantigen genes encoded by the egc cluster and SaPIbov are predominant among Staphylococcus aureus isolates from cows, goats, sheep, rabbits and poultry. J Med Microbiol. 2005;54:401–411. doi: 10.1099/jmm.0.45863-0. [DOI] [PubMed] [Google Scholar]
  3. Bohach G A, Fast D J, Nelson R D, Schlievert P M. Staphylococcal and streptococcal pyrogenic toxins involved in toxic shock syndrome and related illnesses. Crit Rev Microbiol. 1990;17:251–272. doi: 10.3109/10408419009105728. [DOI] [PubMed] [Google Scholar]
  4. McCormick J K, Yarwood J M, Schlievert P M. Toxic shock syndrome and bacterial superantigens: an update. Annu Rev Microbiol. 2001;55:77–104. doi: 10.1146/annurev.micro.55.1.77. [DOI] [PubMed] [Google Scholar]
  5. Bhardwaj N, Young J W, Nisanian A J, Baggers J, Steinman R M. Small amounts of superantigen, when presented on dendritic cells, are sufficient to initiate T cell responses. J Exp Med. 1993;178:633–642. doi: 10.1084/jem.178.2.633. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Dang L H, Michalek M T, Takei F, Benaceraff B, Rock K L. Role of ICAM-1 in antigen presentation demonstrated by ICAM-1 defective mutants. J Immunol. 1990;144:4082–4091. [PubMed] [Google Scholar]
  7. Fischer H, Gjorloff A, Hedlund G, Hedman H, Lundgren E, Kalland T, Sjogren H O, Dohlsten M. Stimulation of human naive and memory T helper cells with bacterial superantigen. Naive CD4+45RA+ T cells require a costimulatory signal mediated through the LFA-1/ICAM-1 pathway. J Immunol. 1992;148:1993–1998. [PubMed] [Google Scholar]
  8. Seo K S, Lee S U, Park Y H, Davis W C, Fox L K, Bohach G A. Long-term staphylococcal enterotoxin C1 exposure induces soluble factor-mediated immunosuppression by bovine CD4+ and CD8+ T cells. Infect Immun. 2007;75:260–269. doi: 10.1128/IAI.01358-06. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Ikejima T, Dinarello C A, Gill D M, Wolff S M. Induction of human interleukin-1 by a product of Staphylococcus aureus associated with toxic shock syndrome. J Clin Invest. 1984;73:1312–1320. doi: 10.1172/JCI111334. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Miethke T, Wahl C, Regele D, Gaus H, Heeg K, Wagner H. Superantigen mediated shock: a cytokine release syndrome. Immunobiology. 1993;189:270–284. doi: 10.1016/S0171-2985(11)80362-1. [DOI] [PubMed] [Google Scholar]
  11. Akdis C A, Blaser K. IL-10-induced anergy in peripheral T cell and reactivation by microenvironmental cytokines: two key steps in specific immunotherapy. FASEB J. 1999;13:603–609. doi: 10.1096/fasebj.13.6.603. [DOI] [PubMed] [Google Scholar]
  12. Sriskandan S, Evans T J, Cohen J. Bacterial superantigen-induced human lymphocyte responses are nitric oxide dependent and mediated by IL-12 and IFN-γ. J Immunol. 1996;156:2430–2435. [PubMed] [Google Scholar]
  13. Tessier P A, Naccache P H, Diener K R, Gladue R P, Neote K S, Clark-Lewis I, McColl S R. Induction of acute inflammation in vivo by staphylococcal superantigens. II. Critical role for chemokines, ICAM-1, and TNF-α. J Immunol. 1998;161:1204–1211. [PubMed] [Google Scholar]
  14. Muraille E, De Trez C, Pajak B, Brait M, Urbain J, Leo O. T cell-dependent maturation of dendritic cells in response to bacterial superantigens. J Immunol. 2002;168:4352–4360. doi: 10.4049/jimmunol.168.9.4352. [DOI] [PubMed] [Google Scholar]
  15. Soltys J, Quinn M T. Selective recruitment of T-cell subsets to the udder during staphylococcal and streptococcal mastitis: analysis of lymphocyte subsets and adhesion molecule expression. Infect Immun. 1999;67:6293–6302. doi: 10.1128/iai.67.12.6293-6302.1999. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Kuroishi T, Komine K, Asai K, Kobayashi J, Watanabe K, Yamaguchi T, Kamata S, Kumagai K. Inflammatory responses of bovine polymorphonuclear neutrophils induced by staphylococcal enterotoxin C via stimulation of mononuclear cells. Clin Diagn Lab Immunol. 2003;10:1011–1018. doi: 10.1128/CDLI.10.6.1011-1018.2003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Deringer J R, Ely R J, Monday S R, Stauffacher C V, Bohach G A. Vβ-dependent stimulation of bovine and human T cells by host-specific staphylococcal enterotoxins. Infect Immun. 1997;65:4048–4054. doi: 10.1128/iai.65.10.4048-4054.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Ackermann M R, DeBey B M, Stabel T J, Gold J H, Register K B, Meehan J T. Distribution of anti-CD68 (EBM11) immunoreactivity in formalin-fixed, paraffin-embedded bovine tissues. Vet Pathol. 1994;31:340–348. doi: 10.1177/030098589403100307. [DOI] [PubMed] [Google Scholar]
  19. Yamakawa Y, Pennelegion C, Willcocks S, Stalker A, Machugh N, Burt D, Coffey T J, Werling D. Identification and functional characterization of a bovine orthologue to DC-SIGN. J Leukoc Biol. 2008;83:1396–1403. doi: 10.1189/jlb.0807523. [DOI] [PubMed] [Google Scholar]
  20. Komohara Y, Hirahara J, Horikawa T, Kawamura K, Kiyota E, Sakashita N, Araki N, Takeya M. AM-3K, an anti-macrophage antibody, recognizes CD163, a molecule associated with an anti-inflammatory macrophage phenotype. J Histochem Cytochem. 2006;54:763–771. doi: 10.1369/jhc.5A6871.2006. [DOI] [PubMed] [Google Scholar]
  21. Wang Z Q, Orlikowsky T, Dudhane A, Trejo V, Dannecker G E, Pernis B, Hoffmann M K. Staphylococcal enterotoxin B-induced T-cell anergy is mediated by regulatory T cells. Immunology. 1998;94:331–339. doi: 10.1046/j.1365-2567.1998.00519.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Kum W W, Cameron S B, Hung R W, Kalyan S, Chow A W. Temporal sequence and kinetics of proinflammatory and anti-inflammatory cytokine secretion induced by toxic shock syndrome toxin 1 in human peripheral blood mononuclear cells. Infect Immun. 2001;69:7544–7549. doi: 10.1128/IAI.69.12.7544-7549.2001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Seo K S, Davis W C, Hamilton M J, Park Y H, Bohach G A. Development of monoclonal antibodies to detect bovine FOXP3 in PBMCs exposed to a staphylococcal superantigen. Vet Immunol Immunopathol. 2008 doi: 10.1016/j.vetimm.2008.10.292. Epub ahead of print. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Tollersrud T, Kampen A H, Kenny K. Staphylococcus aureus enterotoxin D is secreted in milk and stimulates specific antibody responses in cows in the course of experimental intramammary infection. Infect Immun. 2006;74:3507–3512. doi: 10.1128/IAI.01726-05. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Osugi Y, Vuckovic S, Hart D N. Myeloid blood CD11c(+) dendritic cells and monocyte-derived dendritic cells differ in their ability to stimulate T lymphocytes. Blood. 2002;100:2858–2866. doi: 10.1182/blood.V100.8.2858. [DOI] [PubMed] [Google Scholar]
  26. Leon B, Martinez del Hoyo G, Parrillas V, Vargas H H, Sanchez-Mateos P, Longo N, Lopez-Bravo M, Ardavin C. Dendritic cell differentiation potential of mouse monocytes: monocytes represent immediate precursors of CD8– and CD8+ splenic dendritic cells. Blood. 2004;103:2668–2676. doi: 10.1182/blood-2003-01-0286. [DOI] [PubMed] [Google Scholar]
  27. Zou W, Borvak J, Marches F, Wei S, Galanaud P, Emilie D, Curiel T J. Macrophage-derived dendritic cells have strong Th1- polarizing potential mediated by β-chemokines rather than IL-12. J Immunol. 2000;165:4388–4396. doi: 10.4049/jimmunol.165.8.4388. [DOI] [PubMed] [Google Scholar]
  28. Sallusto F, Lanzavecchia A. Efficient presentation of soluble antigen by cultured human dendritic cells is maintained by granulocyte/macrophage colony-stimulating factor plus interleukin 4 and downregulated by tumor necrosis factor α. J Exp Med. 1994;179:1109–1118. doi: 10.1084/jem.179.4.1109. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Zhou L J, Tedder T F. CD14+ blood monocytes can differentiate into functionally mature CD83+ dendritic cells. Proc Natl Acad Sci USA. 1996;93:2588–2592. doi: 10.1073/pnas.93.6.2588. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Vieira P L, de Jong E C, Wierenga E A, Kapsenberg M L, Kalinski P. Development of Th1-inducing capacity in myeloid dendritic cells requires environmental instruction. J Immunol. 2000;164:4507–4512. doi: 10.4049/jimmunol.164.9.4507. [DOI] [PubMed] [Google Scholar]
  31. Stephens S A, Brownlie J, Charleston B, Howard C J. Differences in cytokine synthesis by the sub-populations of dendritic cells from afferent lymph. Immunology. 2003;110:48–57. doi: 10.1046/j.1365-2567.2003.01712.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Bleul C C, Fuhlbrigge R C, Casasnovas J M, Aiuti A, Springer T A. A highly efficacious lymphocyte chemoattractant, stromal cell-derived factor 1 (SDF-1) J Exp Med. 1996;184:1101–1109. doi: 10.1084/jem.184.3.1101. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Dunussi-Joannopoulos K, Zuberek K, Runyon K, Hawley R G, Wong A, Erickson J, Herrmann S, Leonard J P. Efficacious immunomodulatory activity of the chemokine stromal cell-derived factor 1 (SDF- 1): local secretion of SDF-1 at the tumor site serves as T-cell chemoattractant and mediates T-cell-dependent antitumor responses. Blood. 2002;100:1551–1558. [PubMed] [Google Scholar]
  34. Tanaka Y, Adams D H, Hubscher S, Hirano H, Siebenlist U, Shaw S. T-cell adhesion induced by proteoglycan-immobilized cytokine MIP-1 β. Nature. 1993;361:79–82. doi: 10.1038/361079a0. [DOI] [PubMed] [Google Scholar]
  35. Van Damme J, Proost P, Lenaerts J P, Opdenakker G. Structural and functional identification of two human, tumor-derived monocyte chemotactic proteins (MCP-2 and MCP-3) belonging to the chemokine family. J Exp Med. 1992;176:59–65. doi: 10.1084/jem.176.1.59. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Ajuebor M N, Hogaboam C M, Kunkel S L, Proudfoot A E, Wallace J L. The chemokine RANTES is a crucial mediator of the progression from acute to chronic colitis in the rat. J Immunol. 2001;166:552–558. doi: 10.4049/jimmunol.166.1.552. [DOI] [PubMed] [Google Scholar]
  37. Buckley C D, Pilling D, Lord J M, Akbar A N, Scheel-Toellner D, Salmon M. Fibroblasts regulate the switch from acute resolving to chronic persistent inflammation. Trends Immunol. 2001;22:199–204. doi: 10.1016/s1471-4906(01)01863-4. [DOI] [PubMed] [Google Scholar]
  38. Glabinski A R, Bielecki B, Ransohoff R M. Chemokine upregulation follows cytokine expression in chronic relapsing experimental autoimmune encephalomyelitis. Scand J Immunol. 2003;58:81–88. doi: 10.1046/j.1365-3083.2003.01285.x. [DOI] [PubMed] [Google Scholar]
  39. Banisor I, Leist T P, Kalman B. Involvement of β-chemokines in the development of inflammatory demyelination. J Neuroinflammation. 2005;2:7. doi: 10.1186/1742-2094-2-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. De Saint-Vis B, Fugier-Vivier I, Massacrier C, Gaillard C, Vanbervliet B, Ait-Yahia S, Banchereau J, Liu Y J, Lebecque S, Caux C. The cytokine profile expressed by human dendritic cells is dependent on cell subtype and mode of activation. J Immunol. 1998;160:1666–1676. [PubMed] [Google Scholar]
  41. Sallusto F, Palermo B, Lenig D, Miettinen M, Matikainen S, Julkunen I, Forster R, Burgstahler R, Lipp M, Lanzavecchia A. Distinct patterns and kinetics of chemokine production regulate dendritic cell function. Eur J Immunol. 1999;29:1617–1625. doi: 10.1002/(SICI)1521-4141(199905)29:05<1617::AID-IMMU1617>3.0.CO;2-3. [DOI] [PubMed] [Google Scholar]
  42. Mantovani A, Allavena P, Vecchi A, Sozzani S. Regulation of chemokine receptor expression in dendritic cells. Res Immunol. 1998;149:639–641. doi: 10.1016/s0923-2494(99)80030-3. [DOI] [PubMed] [Google Scholar]
  43. Miethke T, Wahl C, Heeg K, Echtenacher B, Krammer P H, Wagner H. T cell-mediated lethal shock triggered in mice by the superantigen staphylococcal enterotoxin B: critical role of tumor necrosis factor. J Exp Med. 1992;175:91–98. doi: 10.1084/jem.175.1.91. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Kuntz M M, Innes J B, Weksler M E. Lymphocyte transformation induced by autologous cells. IV. Human T-lymphocyte proliferation induced by autologous or allogeneic non-T lymphocytes. J Exp Med. 1976;143:1042–1054. doi: 10.1084/jem.143.5.1042. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Opelz G, Kiuchi M, Takasugi M, Terasaki P I. Autologous stimulation of human lymphocyte subpopulation. J Exp Med. 1975;142:1327–1333. doi: 10.1084/jem.142.5.1327. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Scheinecker C, Machold K P, Majdic O, Hocker P, Knapp W, Smolen J S. Initiation of the autologous mixed lymphocyte reaction requires the expression of costimulatory molecules B7–1 and B7–2 on human peripheral blood dendritic cells. J Immunol. 1998;161:3966–3973. [PubMed] [Google Scholar]
  47. Tomai M A, Beachey E H, Majumdar G, Kotb M. Metabolically active antigen presenting cells are required for human T cell proliferation in response to the superantigen streptococcal M protein. FEMS Microbiol Immunol. 1992;4:155–164. doi: 10.1111/j.1574-6968.1992.tb04982.x. [DOI] [PubMed] [Google Scholar]

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