Departments of
* Veterinary Pathobiology, Program for Prevention of Animal Infectious Diseases, and
Molecular Microbiology and Immunology, University of Missouri, Columbia;
Institute of Veterinary Virology, University of Bern, Switzerland; and
Institute for Animal Health, Compton, Newbury, United Kingdom
Correspondence: D. Mark Estes, Department of Veterinary Pathobiology, University of Missouri, 201 Connaway Hall, Columbia, MO 65211. E-mail: EstesD{at}missouri.edu
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Key Words: isotype • immunoglobulin • cytokine • ruminant
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Previous studies from our laboratory have demonstrated a linkage between interleukin (IL)-4 and IL-13 and isotype-restricted responses to immunoglobulin (Ig)G1 and IgE in cattle [14 15 16 17 ]. As bona fide Th1 but not Th2 clones have been identified in cattle, the regulatory requirements necessary to result in biased IgG1 responses in parasite-infected cattle in which this isotype predominates cannot be solely attributed to a completely polarized Th cell population [15 , 16 ]. Given the role of interstitial DC and other DC subpopulations as key players in regulating humoral responses, we sought to determine the potential influence of DC derived from peripheral blood (PB) in regulating the humoral response. This is a key factor in the overall immune physiology of ruminants and cattle, in particular, where selective transport of primarily IgG1 into the colostrum differs from the Ig-isotype profiles of other mammals. Moreover, linkages in effector function inherent within the heavy chain portion of the Ig molecule and resistance to infection have been implied in numerous models of infection involving parasites, bacteria, and viruses [18 19 20 21 22 ]. In this study, we demonstrate that bovine DC derived from PB have the capacity to bias Ig responses toward IgG1, independent of T cell-derived cognate stimulation via expression of functional CD40L transcripts and protein. Thus, cattle have a potential third cellular player that may serve to regulate classical type 2 responses in the absence of a truly polarized T cell subpopulation.
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
(MOPC21, Sigma Chemical Co., St. Louis, MO), mouse IgM, 
(MOPC-104E, Sigma Chemical Co.), and mouse IgG2a, (UPC-10, Sigma Chemical Co.). Cell-bound mAb were detected with fluorescein isothiocyanate (FITC)-conjugated isotype-specific mAb, as indicated: rat anti-mouse IgM (R-60.2), rat anti-mouse IgG2a (R19-15), and rat anti-mouse IgG1 (A85-1). All rat mAb were obtained from PharMingen (San Diego, CA). For detection of bovine (bo)CD80 and CD86, FITC-conjugated goat anti-mouse IgG (H+L, Bethyl Laboratories, Montgomery, TX) was used. For biotinylated CD40-Ig detection of binding to CD40L, we used streptavidin conjugated to phycoerythrin (Becton Dickinson, San Jose, CA). Cells were washed, fixed, and analyzed using a FACS Vantage flow cytometer with CellQuest software (Becton Dickinson).
DC
Blood was collected from healthy Holstein donors in acid citrate dextrose (0.15 M sodium citrate, 80 mM citric acid, 0.16 M dextrose) and centrifuged for 20 min at 400 g to obtain a buffy coat as described previously [23
]. Red cells were removed by lysis. Following washing in Hanks’ balanced saline solution (HBSS; BioWhittaker, Walkersville, MD), CD3+ T cells were removed by negative selection using magnetic beads (sheep anti-mouse-coated beads, Dynal ASA, Oslo, Norway) and mouse antibovine CD3 mAb. T cell-depleted PB mononuclear cells (PBMC) were washed twice, adjusted to 106/ml in complete RPMI [cRPMI; RPMI 1640 supplemented with L-glutamine (Gibco-Life Technologies, Grand Island, NY), 10% fetal bovine serum (Sigma Chemical Co.), and penicillin-streptomycin (Pen-Strep, Sigma Chemical Co.)], and 3 ml of this suspension was added to each well of a six-well plates. Cells were incubated for 2 h, and then nonadherent cells were removed by washing. Adherent cells were then cultured for 6–7 days in cRPMI enriched with growth and differentiation factors: recombinant human granulocyte macrophage-colony stimulating factor (rhGM-CSF; 1400 U/ml; Leukine, Immunex Corp., Seattle, WA), rhFlt-3L (100 ng/ml; R&D Systems, Minneapolis, MN), and rboIL-4 (10 ng/ml or 10% COS cell supernatant). After the initial 3 days of culture, approximately one-half of the medium was removed and replaced with fresh medium and cytokines as indicated above. After 6–7 days of culture, nonadherent cells were collected, resuspended in cRPMI, and isolated over 14.5% metrizamide gradients (Sigma Chemical Co.). In some cases, the metrizamide low-density population of cells after a couple of washes was further cultured overnight in the presence of cytokines to deplete the remaining adherent monocytes. Isolated cells were cytocentrifuged onto glass microscope slides and stained by May-Grunwald staining to visualize morphological features. For phagocytosis assays, DC were exposed for 3 h to FITC-labeled latex-bead particles for 2 h at a 5:1 particle-to-cell ratio. Cells were washed and fixed in 2% buffered paraformaldehyde for fluorescein-activated cell sorter (FACS) analysis.
RNA isolation/preparation
DC were harvested and stimulated with phorbol myristic acid (PMA; 1 ng/ml) and ionomycin (1 µg/ml) in cytokine-enriched (GM-CSF, Flt-3L, and IL-4) cRPMI for 14 h. DC were transferred to RNase-free microcentrifuge tubes and washed twice with HBSS. RNA was extracted using the Qiagen (Valencia, CA) RNeasy mini-kit according to the manufacturer’s protocol. Isolated, total RNA was treated with a DNase treatment and removal kit (Ambion, Austin, TX). Approximately 1.5 mg per treatment was used to synthesize DNA as described previously [23
].
Taqman reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of cytokine transcripts
A total of 1 µl cDNA obtained in the synthesis reaction described above was amplified in each reaction. PCR reactions were performed in 25 µl vol with the following components according to the manufacturer’s protocol: Taqman PCR master mix (Applied Biosystems, Foster City, CA; contains the Amplitaq Gold® DNA polymerase, dNTPs, and buffer), nuclease-free water, forward- and reverse-cytokine primers (IDT, Coralville, IA), 5'(6-carboxy-fluorcein)FAM/3'(6-carboxy-tetramethyl-rhodamine)TAMRA-labeled cytokine probe (Applied Biosystems), 18S ribosomal primers (Perkin-Elmer, Wellesley, MA; forward and reverse), and a 5'VIC/3'TAMRA-labeled ribosomal probe (Applied Biosystems). Reactions were performed in an ABI Prism 7700 sequence detection system with a 48°C incubation for 30 min, a 95°C incubation for 10 min, and 40 cycles of 95°C for 15 s and 60°C for 1 min. The levels of FAM and VIC fluorescence are directly proportional to the amount of cytokine transcripts in the reaction. The relative amounts of steady-state cytokine mRNA present (as compared with the internal standard VIC-labeled ribosomal probe) were calculated according to the following formula: 2-
CT, as described previously [24
]. PCR reactions containing no cDNA template were analyzed in parallel as negative controls.
B cells
B cells were isolated from the same donor as DC for each experiment as described previously [23
]. Briefly, high-density cells (>1.079 g/ml) were enriched over Percoll gradients (Sigma Chemical Co.). Small resting B cells were isolated by positive selection using a mAb reactive with IgM (BM-23; Sigma Chemical Co.) and sheep anti-mouse IgG-coated magnetic beads (Dynal ASA). Cells were routinely >92% positive for surface IgM following this procedure.
Cell culture
A total of 105 surface (s)IgM+ B cells were cultured in 96-well tissue-culture plates with or without DC (105) and/or mitomycin C-treated DAP3 boCD40L-transfected cells (2.5x103) as described previously [25
]. Cells were cultured in RPMI-1640 supplement with 10% normal horse serum (NHS; Gibco-Life Technologies) and antibiotics (Pen-Strep). Cells were cultured in some cases with exogenous cytokine (rhIL-2; R&D Systems) or rh interferon (IFN)-
(PBL Biomedical Laboratories, Newington, NH) as indicated. The levels of secreted Ig subclasses (IgM, IgG1, IgG2, and IgA) were measured in supernatants after 6–7 days of culture by capture enzyme-linked immunosorbent assay (ELISA) as indicated below.
ELISA
Estimation of secretory Ig subclasses was performed as described previously with minor modifications [17
, 25
26
27
]. Briefly, 96-well ELISA plates (Dynatech Immulon II, Chantilly, VA) were coated with 1 µg/well of the respective antibovine-isotype antibody in phosphate-buffered saline (PBS) as follows: goat antibovine IgM (Kirkegaard and Perry Laboratories, Gaithersburg, MD), rabbit antibovine IgA (Bethyl Laboratories), sheep antibovine IgG1 (Bethyl Laboratories), and sheep antibovine IgG2 (Bethyl Laboratories). Plates were incubated overnight at 4°C, washed three times with PBS + 0.05% Tween-20 (Sigma Chemical Co.), and blocked for 1 h at 37°C with 10% NHS. Plates were washed three times as before, and culture supernatants were added at a 1:2 dilution in PBS supplemented with 5% NHS. Plates were incubated for 1 h at 37°C. Following a repeat wash cycle, alkaline phosphatase-conjugated rabbit antibovine IgA (Bethyl Laboratories), alkaline phosphatase-conjugated sheep antibovine IgG1 (Bethyl Laboratories), alkaline phosphatase-conjugated goat antibovine IgM, or alkaline phosphatase-conjugated sheep antibovine IgG2 (Bethyl Laboratories) was added to their respective plates. After a 1-h incubation at 37°C, plates were washed three times and developed with substrate using a commercial kit (Kirkegaard and Perry) at 405 nm. Known standards for each isotype were analyzed in parallel, and concentrations were estimated by linear regression analysis.
boCD40-hIgG1 (Fc)
A hingeless variant of the extracellular domain of the boCD40 homologue to the hIgG1 CH2 and CH3 domains was constructed by an in-frame gene fusion. Briefly, the extracellular domain of boCD40, including its native signal sequence and an added Kozak’s consensus sequence, was fused to cDNA encoding the hIgG1 Fc portion (CH2 and CH3 domains lacking hinge region) and was cloned into the mammalian-expression vector, pcDNA3.1/neo(+; Invitrogen, Carlsbad, CA). CD40-Ig-pcDNA3.1(+) was introduced into MOP8 NIH 3T3 cells (American Type Culture Collection, Manassas, VA; CRL-1709) using Lipofectamine (Gibco-BRL, Gaithersburg, MD) according to the manufacturer’s instructions. Stable transfectants were selected by limited dilution cloning and selection in complete Dulbecco’s modified Eagle’s medium-10 containing 200 µg/ml G418 (Gibco-BRL). Supernatants were analyzed for CD40-Ig via Western blot (data not shown). CD40-Ig was purified from supernatants by dialysis against PBS, pH 7.4, using 50,000 MWCO dialysis tubing cellulose (SpectraPor, Spectrum Laboratories, Inc., Los Angeles, CA). Biotinylated CD40-Ig was found to bind boCD40L transfectants but not the mock-transfected parent cell line (DAP3; murine liver fibroblast cell line; data not shown). Myeloma-derived hIgG1 (Sigma Chemical Co.) was used as a negative control for nonspecific effects of Fc receptor binding.
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
 View larger version (28K): [in a new window] |
Figure 1. Surface phenotype of PB-DC. Nonadherent cells from 7-day cultures were isolated over metrizamide gradients and evaluated by surface staining for various cell-surface molecules. Cells were analyzed by FACS for the markers shown using indirect staining with mouse antibovine-specific mAb (gray line). Isotype controls were run in parallel for the specific isotype of the primary antibody as appropriate (black-shaded areas). Forward- and side-scatter profiles are depicted in the upper left-hand portion of the panel. Histograms were generated and analyzed using Cell Quest software (Becton Dickinson). MFI, Mean fluorescence intensity.
|
and anti-Ig stimulation. B cells cultured under these optimized conditions for IgG2 expression generated similar amounts of this isotype, as we have previously reported (Fig. 3
) [26
]. Donor-to-donor variation has been reported in humans for interdigitating DC induction of IgM and/or IgG/IgA subclasses on coculture [30
]. Our findings are similar to those reported for humans with variation in the capacity of donor-derived DC to induced IgM and/or IgG1.
 View larger version (26K): [in a new window] |
Figure 2. Analysis of secreted Ig isotypes by sIgM+-resting B cells following coculture with DC. Six- to seven-day culture supernatants were analyzed by capture ELISA for Ig-isotype production by sIgM-positive, resting B cells following coculture as described in Materials and Methods. In all experiments, the IgA and IgG2 responses were below the detection limit in our capture ELISA (<20 ng/ml). Control cultures with B cells alone and CD40L-stimulated B cells with or without hIL-2 supplementation (1 ng/ml) were also under the detection limit. Background absorbance was subtracted based on the DC-only culture supernatant. Results are representative of the mean and standard deviation of the mean for triplicate cultures for each experiment. Relative amounts of secretory Ig produced were determined by linear regression versus a known standard for individual isotypes. The data shown are from a total of eight experiments. (A) DC and B cells derived from donor 1; (B) DC and B cells from donor 2.
|
 View larger version (9K): [in a new window] |
Figure 3. Resting sIgM+ B cells have the potential to switch to IgG2 with or without DC following B cell receptor cross-linking and coculture with rhIFN-. Six- to seven-day culture supernatants were analyzed by capture ELISA for Ig-isotype production by sIgM-positive resting B cells following coculture as described in Materials and Methods. Results are shown for the absorbance at 405 nm following capture ELISA for the IgG2 isotype from B cell culture supernatants from a common donor used in the experiments presented in Figures 2
and 4
. Cells were treated as indicated. Results are representative of the mean and standard deviation of the mean for triplicate cultures for each experiment.
|
(TNF-). Also detected but expressed at relatively low levels were transcripts for IL-6, IL-10, IL12p40, IL-13, and CD40L. DC were stimulated with phorbol ester to further demonstrate their potential for cytokine production. Following this treatment, DC expressed increased transcripts for IL-1ß (approximately a 13-fold increase vs. untreated cells), IL-6 (>3000-fold), IL-10 (
200-fold), IL12p40 (90,000-fold), TNF- (16-fold), IL-4 (13,000-fold), IL-13 (>500-fold), and CD40L (2800-fold). Collectively, these results suggest that DC have the potential to produce a mixed profile of proinflammatory, inflammatory, and type 2 regulatory cytokines to up-regulate costimulatory molecules and potentially bias humoral responses toward IgG1 production. |
View this table: [in a new window] |
Table 1. Relative Quantification by Taqman RT-PCR Analysis of Steady-State Cytokine mRNA in Stimulated and Unstimulated PB-DC
|
 View larger version (15K): [in a new window] |
Figure 4. CD40-Ig blocks CD40-dependent induction of IgM by DC in coculture with IgM+-resting B cells. Naive B cells (105) were cultured with PB-DC (105) in the presence of rhIL-2 (1 ng/ml) with or without CD40-Ig fusion protein added (final concentration, 2 µg/ml). As a negative control for effects of the hIgG1 Fc, hIgG1 (2 µg/ml) was added to DC-B cell culture. After 6–7 days of coculture, supernatants were collected and analyzed by capture ELISA for IgM secretion. Results are representative of the mean and standard deviation of the mean for triplicate cultures for each variable. Data are representative of two independent experiments from different donors. Relative amounts of secreted IgM were determined by linear regresion versus a known standard.
|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
production [15
, 16
]. As classical Th2 clones have not been described in cattle, to our knowledge, this suggests that other cell types or alternatively, IL-13 may play larger roles in initiating type 2 responses in a species with the potential for a type 1 bias. Despite the fact that DC may fill this role in vivo, it might also be possible that no DC1-DC2 system and hence, no clear-cut Th1-Th2 bias exist in cattle. Given the potential to express CD40L and thus generate signals via CD40, DC have the repertoire available to augment IgG1 over IgG2 responses in anatomic locations where they may come into physical contact with resident or recirculating B cells. This observation is consistent with other mammalian species, as hDC have been shown to express functional CD40L (CD154) on their cell surface [31
]. DC also serve a pivotal role in priming naïve T cells and may also impact the functional activities of follicular T cells within the B cell areas of lymphoid tissues. These interactions remain to be fully elucidated in cattle. In humans, CD40 engagement is critical for the induction of isotype switching. This requirement is best exemplified in hyper-IgM syndrome, in which a genetic defect in the CD40L gene results in a deficit of circulating IgG and IgA and germinal center formation. hDC have been shown to promote the expansion and differentiation of CD40-activated B cells [35 , 36 ]. In humans, IL-4 and IL-13 promote isotype switching to IgE and IgG4, the isotype equivalent in terms of cytokine regulation to bovine IgE and IgG1, respectively [14 , 17 , 37 ]. hIL-10 induces class switching in human B cells to IgG1, IgG3, and IgA [38 39 40 ] but does not appear to act as a switch factor in cattle. However, IL-10 does appear to be an important cofactor for IgA production by bovine B cells under certain activation conditions in vitro [26 ]. Thus, the findings presented in the present study would suggest that DC in cattle, via interactions with B cells and other cell types including T cells, have the potential to generate multiple antibody types including IgG1 and through cofactor production, IgA. Thus, DC may aid to overcome a potential deficit in T cell cognate interactions and the production of soluble factors as reflected in the cytokine repertoire where the Th0 or Th1 cell predominates. In the context of the recently described DC1-DC2 system, it would be interesting to examine whether the increase in IL-13 production by the DC generated in the present study can affect Th2 development, as described recently for the murine system [41 ]. Th2 development has been demonstrated to be impaired in IL-13 knockout mice [42 ]. IL-13 is clearly a key player in allergic asthma and nematode infections [43 , 44 ]. Thus, IL-13-producing DC may play an important role in several of these diseases and clearly merit further study and may support the switch of bovine B cells toward multiple antibody types including IgG1 and IgE.
Received March 13, 2002; accepted October 2, 2002.
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
This article has been cited by other articles:
 |
|
 |
|
  A. S. Austin, K. M. Haas, S. M. Naugler, A. A. Bajer, D. Garcia-Tapia, and D. M. Estes Identification and Characterization of a Novel Regulatory Factor: IgA-Inducing Protein J. Immunol., August 1, 2003; 171(3): 1336 - 1342. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
