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Sections of
*
Atherosclerosis and
Pulmonary and Critical Care Medicine, Department of Medicine,
Department of Immunology, and
Section of Leukocyte Biology, Department of Pediatrics, Baylor College of Medicine, Houston, TX 77030; and
¶ ICOS Corp., Bothell, WA 98021
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Abstract |
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2 integrin CD11a is involved in T cell-APC interactions, but the roles of CD11b, CD11c, and CD11d in such interactions have not been examined. To evaluate the roles of each CD11/CD18 integrin in T cell-APC interactions, we tested the ability of splenocytes of CD11-knockout (KO) mice to respond to staphylococcal enterotoxins (SEs), a commonly used superantigen. The defect in T cell proliferation with SEA was more severe in splenocytes from mice deficient in CD18, CD11b, or CD11d than in CD11a-deficient splenocytes, with a normal response in CD11c-deficient splenocytes. Mixing experiments showed that the defect of both CD11b-KO and CD11d-KO splenocytes was, unexpectedly, in T cells rather than in APC. Cytometric analysis failed to detect CD11b or CD11d on resting or activated T cells or on thymocytes of wild-type adult mice, nor did Abs directed to these integrins block responses in culture, suggesting that T cells educated in CD11b-KO or CD11d-KO mice were phenotypically altered. Consistent with this hypothesis, T cells from CD11b-KO and CD11d-KO splenocytes exhibited reduced intensity of CD3 and CD28 expression and decreased ratios of CD4/CD8 cells, and CD4+ T cells were reduced among CD11b-KO and CD11d-KO thymocytes. CD11b and CD11d were coexpressed on a subset of early wild-type fetal thymocytes. We postulate that transient thymocyte expression of both CD11b and CD11d is nonredundantly required for normal thymocyte and T cell development, leading to phenotypic changes in T cells that result in the reduced response to SE stimulation. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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subunits (CD11a–d), with a common 2 chain (CD18) (1). CD11/CD18 integrins play a critical role in leukocyte adhesion by binding to a number of receptors such as ICAM-1, ICAM-2, and ICAM-3, members of the Ig gene superfamily, and other proteins such as inactivated C3b and fibrinogen (1, 2). Patients with leukocyte adhesion deficiency type I (LAD I)3 have mutations in CD18 that lead to a severe or total deficiency of the CD11/CD18 integrins from the cell surface (3, 4). Although the genetic disorder LAD I has given great insight into the functional significance of the CD11/CD18 family, the relative contributions of each of the CD11 integrins in the phenotypic abnormalities seen in LAD I are poorly understood. Mice with a null mutation for CD18 do not express CD11/CD18 integrins and exhibit marked defects in neutrophil adhesion, migration, and host defense similar to humans with LAD I (2). CD18-null mice also show severe defects in T cell proliferation in response to staphylococcal enterotoxin (SE) A, suggesting that CD11/CD18 integrins play a critical role in T cell- and/or APC-dependent immune response. Because Abs blocking CD11a and ICAM-1 inhibit SE-induced T cell proliferation and cytokine production, the involvement of CD11a/CD18 has been previously suggested. However, as shown later in this report, a defect in T cell proliferation in response to SE was more severe in CD18-null mice than CD11a-null mice, suggesting a role of other CD11 integrins. In addition to their functions as adhesive ligands, CD11/CD18 integrins also play an important role in cell signaling. Previous work in our laboratory has shown that canine neutrophils use CD11a and CD11b to interact with ICAM-1 and signal H2O2 production, with CD11a at an early stage not requiring prior chemotactic stimulation and CD11b at a later stage requiring chemotactic stimulation (5).
SEs, the most widely studied superantigens, which include SEA, SEB, SEC, SED, and SEE, stimulate proliferation of subsets of T cells by directly cross-linking the variable region of certain TCR -chains with MHC class II molecules on the surface of APC (6, 7). In SE-induced T cell proliferation, the interaction between SE/MHC-II on APC with TCR on T cells is commonly referred to as signal 1 (8). SE-induced T cell proliferation also requires a signal 2, which involves the interactions of CD2, CD28, and CD11a on T cells with their respective ligands CD58 (LFA-3), B7/BB1, and ICAM-1 on APC (8, 9, 10).
In the present study, we evaluated the role of CD11a, CD11b, CD11c, and CD11d in the response of splenocytes to SE stimulation, by using mice deficient in each CD11 integrin developed by our laboratory.
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Materials and Methods |
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A genomic 
phage clone containing an 8.3-kb fragment of the mouse CD11d gene was isolated from an SV129 murine genomic library and used to develop a construct for targeted homologous recombination as shown in Fig. 1. A 4.1-kb genomic restriction fragment located upstream of exon 1 and a 1.7-kb genomic restriction fragment located downstream of exon 2 were ligated into the polylinker of pBluescript SK+ (Stratagene, La Jolla, CA). A neomycin cassette driven by the mouse RNA polymerase II promoter was inserted between the two fragments (Fig. 1b). All ligations were confirmed by DNA sequencing and restriction digests. The construct was linearized by digestion with NotI, which cleaves in the polylinker. The construct was designed so that targeted homologous recombination with the murine CD11d gene would result in the replacement of a 2.2-kb genomic fragment containing exons 1 and 2 with the 1.8-kb neomycin cassette (Fig. 1c).
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CD11a-, CD11b-, and CD11c-deficient mice and animal experiments
CD11a-knockout (KO) and CD11b-KO mice were generated in our laboratory as previously described (13, 14), and CD11c-KO mice were also developed in our laboratory by targeted homologous recombination techniques (H. Wu, H. Wang, J. L. Perrard, X.-Y. D. Perrard, Y. Abe, A. R. Burns, D. C. Bullard, C. W. Smith, and C. M. Ballantyne, manuscript in preparation). All CD11-KO mice were backcrossed for at least six generations onto C57BL/6 background (Harlan Sprague Dawley, Indianapolis, IN), and C57BL/6 mice purchased from Harlan Sprague Dawley were used as wild-type (WT) controls. For each experiment, age- and sex-matched CD11-KO mice and WT mice were used. All mice were housed in autoclaved microisolator cages, with autoclaved feed. All animal studies were approved by the Animal Protocol Review Committee of Baylor College of Medicine.
Preparation of mouse splenocytes and thymocytes
Mouse splenocytes and thymocytes were prepared as described by Kruisbeek (15), with some modifications. Briefly, after the mice were sacrificed under anesthesia, fresh spleens and/or thymuses were removed. Fetal thymuses were dissected from mouse fetuses under a dissecting microscope on the days indicated in the result section. The day on which vaginal plug occurred was counted as day 0 of gestation. The mouse spleens or thymuses were minced with a sharp sterile blade, placed in a 40-µm nylon cell strainer (BD Biosciences, Franklin Lakes, NJ), and pressed with the plunger of a 6-ml syringe until mostly fibrous tissue remained. The splenocyte or thymocyte suspension in RPMI 1640 supplemented with 5% FBS was collected. The red blood cells were lysed with lysing buffer (0.15 M NH4Cl, 10.0 mM KHCO3, 0.1 mM Na2EDTA, pH 7.4), and the dead cells were removed by a one-step gradient method (15). After being washed once with RPMI 1640 with 5% FBS, the mouse splenocytes or thymocytes were resuspended in RPMI 1640 with 10% FBS.
Purification of T cells from mouse splenocytes
T cells were purified from mouse splenocytes by depletion of non-T cells (negative selection) with a Pan T cell isolation kit (Miltenyi Biotec, Bergisch Gladbach, Germany). First, mouse splenocytes were stained with biotin-Ab mixture, which includes biotin-conjugated mAbs against mouse CD11b, CD45R, DX5, and Ter-119. Then, the biotin Ab-stained mouse splenocytes were coincubated with anti-biotin microbeads. Finally, purified T cells were obtained by magnetic separation. For T cell purification from CD11b-KO mouse splenocytes, the combination of the Pan T cell isolation kit with mouse MHC class II microbeads was introduced.
In vitro T cell proliferation upon SE stimulation
For the detection of in vitro T cell proliferation in whole splenocytes, whole mouse splenocytes were plated in 96-well round-bottom microtiter plates at 4 x 105 cells/well in a total volume of 200 µl of RPMI 1640 supplemented with 10% FBS, 100 U/ml penicillin, 100 µg/ml streptomycin, and 50 µM 2-ME. The cells were cultured in the absence (control) or presence of SEs (Toxin Technology, Sarasota, FL) for 72 h, then labeled with 1 µCi of [3H]thymidine (DuPont-New England Nuclear, Boston, MA) per well for an additional 18 h. After that, the cells were harvested and counted by liquid scintillation spectroscopy (Betaplate; Wallace, Gaithersburg, MD). The results were expressed as mean counts per minute ± SEM of triplicate cultures. Mouse splenocytes were also stimulated with Con A, PHA, and PMA-ionomycin (Sigma-Aldrich, St. Louis, MO). For the Ab-blocking experiment, WT splenocytes were incubated at 4°C for 30 min with 20 µg/ml anti-mouse CD11b (M1/70, rat IgG2b; BD PharMingen, San Diego, CA), CD11d (205C, hamster IgG; ICOS, Bothell, WA), or CD18 (GAME-46, rat IgG1; BD PharMingen) Abs and then stimulated with SEA for 72 h at 37°C as described above. Rat IgG2b, hamster IgG, and rat IgG1 (BD PharMingen) were used as isotype controls.
For the assessment of purified T cell proliferation, mixing experiments were performed in which purified T cells were coincubated with APC and stimulated with SEs. Purified T cells were obtained from mouse splenocytes as described above. Mouse splenocytes irradiated to 1500 rads were used as APC. Purified T cells (1.3 x 105/well) and irradiated splenocytes (2.6 x 105/well) were cultured in 200 µl of the medium described above in the absence or presence of SEs for 72 h, labeled with [3H]thymidine for another 18 h, and counted by liquid scintillation spectroscopy as described above.
In vivo T cell responses to SEA stimulation in mice
For the in vivo study, mice were injected i.p. with SEA (100 µg dissolved in 0.2 ml saline for each mouse, with saline only as control), and the responses were compared between CD11-KO mice and WT mice matched for age (8–12 wk old) and sex (both male and female) as described by Marrack et al. (16). Briefly, mice were confined, two to three per cage, for 2 wk before beginning the experiment to allow social relationships to stabilize in the group. To observe the in vivo effect of SEA on T cell responses, mice were sacrificed under anesthesia on day 4 after SEA (or saline as control) administration. The thymuses and spleens were removed for cellularity assessment. Differences in average cell numbers were calculated as average splenocyte (thymocyte) numbers of five SEA-inoculated mice/average splenocyte (thymocyte) numbers of five control mice.
Abs and flow cytometric analysis
The following Abs to mouse Ags were used: CD11b (M1/70, FITC, or PE-conjugated); CD11d (205C); CD3 (17A2, PE-conjugated; BD PharMingen); CD4 (H129.19, rat IgG2a, FITC-conjugated; BD PharMingen); CD8 (53-6.7, rat IgG2a, PerCP-conjugated; BD PharMingen); CD28 (37.51, hamster IgG2, PE-conjugated; BD PharMingen); and CD117 (ACK45, rat IgG2b, PE-conjugated; BD PharMingen). FITC-conjugated goat anti-hamster IgG was purchased from Jackson ImmunoResearch Laboratories (West Grove, PA).
For flow cytometric analysis, cells (splenocytes or thymocytes, 1 x 106 in a volume of 100 µl of PBS) were stained in one step with one or two of the above fluorochrome-labeled Abs (1 µg), except for CD11d, which was labeled in two steps with unconjugated hamster anti-CD11d Ab for the first step and FITC-conjugated goat anti-hamster IgG for the second step. After incubation with the indicated Abs for 15 min on ice, the cells were washed three times with cold PBS and finally resuspended in 400 µl of PBS with 1% paraformaldehyde. Data were collected with a FACScan and analyzed with CellQuest software (BD Biosciences, San Jose, CA).
Assessment of NK cell activity in vitro
The mouse lymphoma cell line YAC, maintained in MEM with 100 µg/ml gentamicin, 2 mM L-glutamine, and 5 mM HEPES, was used as the target. The effectors were splenocytes harvested from mice that received i.p. injections of 100 µg of polyinosinate-polycytidylate (for NK priming) the day before. Target cell lysis was assayed by 51Cr release. YAC cells were incubated with Na51CrO4 for 1 h to internalize the 51Cr. After washing and gradient purification, the 51Cr-YAC cells were plated in triplicate in round-bottom 96-well culture plates (5000 cells/well) and coincubated with the mouse splenocytes (effector) at the E:T ratios indicated in Results in a total volume of 200 µl of medium for 4 h at 37°C. Cytotoxicity was determined by the release of 51Cr into the supernatant. Spontaneous release was defined as 51Cr release by target cells in the absence of effector, and maximum release was determined by target cell lysis with SDS. Percent specific lysis was calculated as: specific lysis (%) = [(experimental release – spontaneous release)/(maximum release – spontaneous release)] x100.
Statistics
Results are expressed as mean ± SEM. Statistical comparisons were made by two-tailed unpaired Student’s t test or two-way ANOVA as appropriate. Differences are considered significant at p < 0.05.
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Results |
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ES cells with a targeted event had a 3.5-kb XbaI fragment identified by the 3'-flanking probe on Southern blot compared with the 8.3-kb fragment in WT 129/Sv mice (Fig. 1d). Targeted ES cell clones were injected into C57BL/6 blastocysts, and male chimeric offspring that were >90% agouti coat color were bred to C57BL/6 mice. Germline transmission was confirmed by Southern blotting studies. Mice heterozygous for the mutation were cross-bred to generate homozygous mice. The expected targeted allele of 3.5 kb was identified by the 3'-flanking probe on Southern blots of DNA from mice carrying the mutation (Fig. 1e).
Homozygous and heterozygous mutant mice were born in expected ratios and were fertile. CD11d mutant mice did not demonstrate any gross abnormalities in growth and development and did not develop an increased rate of spontaneous infections when maintained in autoclaved microisolator cages. CD11d mutant mice had normal peripheral leukocyte counts and the same differentials as WT mice. The basal IgG levels in the serum of CD11d mutant mice were normal (data not shown). Two-color flow cytometric analysis showed that in WT mice, CD11d was predominantly expressed on CD11b-positive cells in splenocytes, and no detectable CD11d expression was found on WT peripheral leukocytes. In CD11d mutant mice, there was no detectable expression of CD11d on either splenocytes or peripheral leukocytes.
SEA-induced T cell proliferation in splenocytes deficient in CD11d and other CD11/CD18 integrins in vitro
On the basis of the previous Ab-blocking and transfection studies that showed the involvement of CD11a-ICAM-1 interaction in superantigen-induced lymphocyte proliferation, we first detected SEA-induced T cell proliferation in whole splenocytes from the four CD11(a–d)-KO and CD18-KO mice. As shown in Fig. 2, on in vitro SEA stimulation (0–200 pg/ml), T cell proliferation in CD11a-deficient splenocytes was markedly reduced compared with that in WT mice, which was consistent with previous Ab-blocking studies. At the same time, CD18-deficient splenocytes showed a more severe defect in SEA-induced T cell proliferation, whereas CD11b- and CD11d-deficient splenocytes showed almost the same severe defect in SEA-induced T cell proliferation as CD18-deficient splenocytes. At low concentrations (0–200 pg/ml), SEA induced little T cell proliferative response, with <10,000 cpm of [3H]thymidine incorporation in CD11b-, CD11d-, and CD18-KO splenocytes, compared with high level T cell proliferation with up to >300,000 cpm of [3H]thymidine incorporation in WT splenocytes (Fig. 2). In contrast, CD11c-deficient mice had normal in vitro T cell-proliferative response to SEA (Fig. 2). These results indicated that functional genes for both CD11b and CD11d are required nonredundantly for superantigen responses. To test the hypothesis that splenocytes must express CD11b or CD11d at some time during the culture period, we cultured splenocytes separately with Abs targeting CD18, CD11b, or CD11d. As expected, anti-CD18 significantly inhibited T cell proliferation. However, neither anti-CD11d nor anti-CD11b Ab inhibited SEA-induced T cell proliferation in WT splenocytes in vitro (data not shown).
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Among the four types of CD11-deficient mice, CD11b- and CD11d-deficient splenocytes showed the poorest in vitro T cell response to SEA stimulation (Fig. 2); thus, our studies focused on these two mutations. On the basis of the difference in TCR V specificity among SEB, SEE, and SEA (7, 17), we then determined T cell proliferation in CD11b- and CD11d-deficient splenocytes in response to SEB, SEE, and SEA at a wider range of concentrations. As shown in Fig. 3, compared with WT mice, both CD11b- and CD11d-KO mice had similar defects in T cell proliferative response to SEA, SEB, and SEE stimulation. CD11b- and CD11d-KO splenocytes did respond to high concentrations (>1.0 ng/ml SEA and SEB and >10 ng/ml SEE) of SEs. The T cell response in the KO splenocytes approached the maximum level for WT splenocytes (up to >300,000 cpm of [3H]thymidine incorporation) after stimulation with 100 ng/ml SEs (Fig. 3).
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Lectins such as PHA and Con A may activate T cells by indirectly cross-linking the TCR. In comparison with those of WT mice, splenocytes from CD11b- and CD11d-KO mice responded poorly to PHA and Con A stimulation (Fig. 4, a and b). When splenocytes were stimulated with PMA-ionomycin, CD11b- and CD11d-deficient mice did not show defects in the proliferative response as compared with WT mice (Fig. 4c), suggesting that T cells from CD11b- and CD11d-deficient mice were able to proliferate normally if TCR was bypassed.
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In mouse splenocytes, both T cells and APC are required for the normal response to SEs. To assess which cells (T cells or APC or both) in CD11b- and CD11d-deficient splenocytes are responsible for the defective proliferative response to SEs, we mixed purified T cells with APC and stimulated with SEA. As expected, purified T cells or APC by themselves did not respond to SEA. When purified T cells from WT splenocytes were coincubated with WT APC, SEA induced high level T cell proliferative responses, with a maximum of >300,000 cpm (Fig. 5a). When coincubated with CD11b- and CD11d-deficient APC, WT purified T cells proliferated normally in response to SEA stimulation, which indicated that CD11b- and CD11d-deficient APC were able to present SEA normally. When coincubated with WT APC, however, purified T cells from both CD11b- and CD11d-KO splenocytes exhibited markedly reduced response to SEA (Fig. 5, b and c), which suggested that T cells rather than APC in CD11b- and CD11d-deficient mice contributed to the defective response to SE stimulation.
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To determine the in vivo proliferative responses of CD11b- and CD11d-KO mice to SEA stimulation, CD11b- and CD11d-KO mice and WT mice were injected i.p. with 100 µg of SEA per mouse. After SEA injection, spleen and thymus cellularity from the inoculated mice was measured. As shown in Fig. 6a, SEA injection induced a marked increase in the numbers of spleen cells in WT mice (2.7 times greater average splenocyte numbers for 5 SEA-inoculated mice relative to those for control mice), but a smaller increase in the numbers of spleen cells in CD11b-KO and CD11d-KO mice (1.4 times greater for CD11b-KO and CD11d-KO mice; p < 0.01 compared with WT). In vivo SEA injection, however, as reported previously (16), induced thymus depletion in mice as indicated by a substantial decrease in thymocyte numbers from SEA-treated mice as compared with saline-injected controls. Compared with WT mice (in which the ratio of the average thymocyte numbers of 5 SEA-treated mice relative to those of control mice was 0.31), CD11b-KO and CD11d-KO mice showed less of a decrease in the numbers of thymocytes (thymus depletion) when injected with SEA (ratios of 0.64 and 0.6 for CD11b-KO and CD11d-KO mice, respectively, p < 0.01 compared with WT; Fig. 6b).
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As described above, CD11b- and CD11d-deficient splenocytes showed reduced proliferative responses to SE stimulation both in vitro and in vivo, and the in vitro data indicated that the defects in CD11b- and CD11d-deficient splenocytes were due to T cells rather than APC. Therefore, we assessed the expression of CD11b and CD11d on WT T cells by two-color flow cytometry using CD3 as the specific marker for T cells. At baseline (without SE stimulation), few T cells (CD3-positive cells) in WT splenocytes expressed CD11b or CD11d. All the CD11b- and CD11d-positive cells were CD3 negative (data not shown), i.e., at baseline, there was no detected expression of CD11b and CD11d on WT T cells, and both CD11b and CD11d were expressed on non-T cells in WT splenocytes. We then determined CD11b and CD11d expression on WT T cells after SEA stimulation. Similar to baseline, stimulation of WT splenocytes with SEA for 3 days did not significantly enhance the expression of CD11b or CD11d on WT T cells (data not shown).
Phenotyping of T cells from CD11b- and CD11d-deficient mice
Because there was no expression of CD11b or CD11d detected on WT T cells at baseline and SE stimulation did not enhance expression of CD11b or CD11d, the defective responses of CD11b- and CD11d-deficient mouse T cells to SEs were probably not due to the absence of CD11b or CD11d from T cells per se. We therefore investigated whether the absence of CD11b or CD11d may have caused phenotypic changes in splenocytes or thymocytes that may contribute to the decreased response to SE stimulation. Based on the important roles of CD3 and CD28 in superantigen-induced T cell proliferation (8), we first examined CD3 and CD28 expression on mouse splenocytes by flow cytometry. The percentages of CD3- and CD28-positive cells in CD11b- and CD11d-deficient splenocytes were not different from those of WT splenocytes, which indicated that the T cell percentages in CD11b- and CD11d-deficient whole splenocytes were similar to those in WT splenocytes. As shown in Fig. 7, a and b, however, splenocytes from CD11b-deficient and CD11d-deficient mice had reduced CD3 (with CD3 mean fluorescence intensity (MFI) of 255 for CD11b-deficient and 216 for CD11d-deficient splenocytes) and CD28 (with CD28 MFI of 21 for both CD11b-deficient and CD11d-deficient splenocytes) expression compared with WT splenocytes (with CD3 and CD28 MFI of 366 and 29, respectively). In contrast, CD11a-deficient splenocytes did not show any difference in the intensity of CD3 (with MFI of 379) and CD28 (with MFI of 28) expression compared with WT splenocytes (Fig. 7, a and b). Because T cells mature in the thymus, we next assessed the expression of CD3 and CD28 on mouse thymocytes. As illustrated in Fig. 7c, CD3 expression on CD11b-deficient and CD11d-deficient thymocytes was also reduced (with CD3 MFI on single-positive cells of 574 for CD11b-deficient and 549 for CD11d-deficient thymocytes, compared with 903 for WT). Because a previous study showed that a mAb to CD4 Ag markedly inhibited superantigen-induced T cell proliferation (8), we then measured CD4 and CD8 expression on mouse splenocytes and thymocytes by two-color flow cytometry, and the CD4-positive:CD8-positive cell ratios were calculated (Table I). The ratios of CD4-positive to CD8-positive cells were significantly lower in both CD11b- and CD11d-deficient splenocytes and thymocytes than in WT mice.
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There was no detectable CD11b or CD11d expression on WT splenocyte T cells, but T cells from CD11b- and CD11d-deficient splenocytes and thymocytes did show other phenotypic changes. Therefore, the expression of CD11b and CD11d on mouse thymocytes was evaluated. No CD11b or CD11d expression was found on adult (6 wk old) WT (Fig. 8, a and b) or newborn (2 days old) WT thymocytes (data not shown). However, as shown in Fig. 8c, 
41.5% of day 12 WT fetal thymocytes were CD11b positive, and the majority of the C-kit-positive population was CD11b positive. At this time point in thymic development, >70% of CD11b-positive cells were C-kit positive. Approximately 13% of the day 12 WT fetal thymocytes were CD11d positive (Fig. 8d), and most of the CD11d-positive cells at this stage were also CD11b positive (Fig. 8e). Analysis of later stage WT fetal thymocytes showed that the expression of CD11b and CD11d on the fetal thymocytes fell rapidly. The percentages of CD11b-positive cells in fetal thymocytes decreased from 41.5% on day 12, to 9% on day 13, to 4% on day 14, and to 0.9% on day 17 of gestation (data not shown). In contrast to those on day 12, the majority of CD11b-positive cells in later stage fetal thymocytes were C-kit negative, although the percentage of C-kit-positive cells in mouse fetal thymocytes was not markedly reduced until day 14 of gestation. The percentages of CD11d-positive cells in fetal thymocytes decreased from 13% on day 12, to 4% on day 13, and to <1% on day 14 and day 17 of gestation (data not shown).
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The cytotoxicity of the NK cells from CD11(a–d)-KO mouse splenocytes was also determined. Using YAC as the target, and polyinosinate-polycytidylate-induced splenocytes as effectors, we found that NK cytotoxicity was reduced in CD11a-KO mice compared with WT mice (Fig. 9), consistent with previous studies (18, 19, 20, 21). At the same time, no difference was found in NK cell cytotoxicity in CD11b-KO, CD11c-KO, and CD11d-KO mice compared with WT mice (Fig. 9).
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Discussion |
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Splenocytes from mice deficient in CD18 have been demonstrated to have a marked defect in T cell proliferation to SEA, and this was postulated to be due to the absence of CD11a, which is the ligand for ICAM-1 (2). Abs blocking CD18, CD11a, or ICAM-1 each inhibited SE-induced T cell growth and cytokine production (9, 29, 30, 31). Additionally, transfected or purified ICAM-1 restored SE-induced T cell proliferation via CD11a binding. Naive or resting T cells were particularly dependent on CD11a stimulation (2, 10). Our present study confirmed the role of CD11a in SEA-induced T cell response as indicated by a defect in T cell proliferation in CD11a-deficient splenocytes stimulated with low dose SEA in vitro. Our new finding was that the defect in SEA-induced T cell proliferation was more severe in CD18-, CD11b-, or CD11d-deficient splenocytes than in CD11a-deficient splenocytes in vitro. The in vivo study showing a smaller increase in the numbers of spleen cells in CD11b-KO and CD11d-KO mice than in WT controls after SEA inoculation confirmed the in vitro finding. Thus, among the CD11/CD18 integrins, CD11b and CD11d, in addition to CD11a, contribute to the proliferative response to SEA stimulation. The reduced thymus depletion in CD11b-KO and CD11d-KO mice after SEA inoculation in vivo may also reflect the reduced T cell activation in these KO mice, because the in vivo pathological effects of SE on mice may be T cell mediated (16).
Each SE reacts with T cells bearing the particular V sequences of TCR, inducing activation of these particular populations of T cells (7, 17). The V specificities of SEA for mouse T cells are 1, 3, 10, 11, 17, whereas the specificities of SEB and SEE are 3, 7, 8.1–8.3, 17, and 11, 15, 17, respectively (17). CD11b- and CD11d-deficient splenocytes showed defects in SEB- and SEE-induced T cell proliferation similar to the defects in the SEA-induced T cell response, indicating that the defects do not result from changes in V subsets. The T cell response to high dose SEs and normal T cell proliferation in response to PMA-ionomycin observed in CD11b- and CD11d-deficient splenocytes indicate that T cells themselves in CD11b-KO and CD11d-KO mice have the ability to proliferate, and they are able to proliferate normally if TCR and costimulatory signals are bypassed.
Both T cells and APC in mouse splenocytes are required for the normal response to SE stimulation. The interaction between CD11a on T cells and ICAM-1 on APC is considered to be essential for the normal response to SE stimulation (29, 30, 31). CD11a is expressed predominantly on T cells and serves there as a costimulatory molecule for T cell activation (32). Our mixing experiments showed that T cells, rather than APC, in CD11b- and CD11d-deficient splenocytes had the defect that accounted for the reduced response to SE stimulation. This was unexpected, because neither CD11b nor CD11d has been shown to be predominantly expressed on WT T cells. CD11b is considered to be expressed predominantly on monocytes/macrophages, PMN, and NK cells (33, 34, 35), whereas CD11d is expressed mainly on subsets of the myelomonocytic lineage (myeloid cells), particularly on monocytes, macrophage foam cells, and splenic red pulp macrophages (36, 37, 38, 39, 40).
In humans, CD11d is expressed on peripheral leukocytes, including neutrophils, eosinophils (especially upon stimulation with PMA or fMet-Leu-Phe), monocytes, and to a lesser extent, lymphocytes (38, 39). In contrast to human leukocytes, no detectable CD11d expression was found on WT mouse peripheral leukocytes in our current study. In humans, 2–10% of T cells (mainly CD8-positive cells) in human peripheral blood express CD11b (35, 41). Brief exposure of resting T cells (both CD4 and CD8) to phorbol ester resulted in an increase of CD11b on the surface of a small subpopulation of human T cells (41). Abs to CD11b inhibited anti-CD3-induced human T cell proliferation as well as IL-2 release (35). In contrast, our two-color flow cytometric analysis of mouse splenocytes showed that few T cells in WT splenocytes expressed CD11b or CD11d at baseline or after SE stimulation, which rules out the possibility that the defect in SE-induced T cell proliferation in CD11b- and CD11d-deficient mice is due to the absence of CD11b and CD11d in responding mouse T cells.
As previously reported, interference with the SE/MHC-CD3/TCR complex (signal 1) by using mAbs to CD3 or MHC class II strongly inhibited T cell proliferation induced by low doses of superantigen (8). Blocking the CD28 costimulatory pathway (signal 2) with an anti-CD28 mAb also achieved similar inhibition of T cell response to superantigen stimulation (8, 9, 29, 30, 31). These studies suggest that each of the molecules makes a major contribution to T cell activation by MHC-superantigen complex stimulation. As an investigation of the defects in T cells of CD11b- and CD11d-KO mice that contribute to the decreased response to SE stimulation, we found reduced intensities of CD3 and CD28 expression on CD11b- and CD11d-deficient splenocytes as compared with those of WT mice. Based on the important roles of CD3 and CD28 in SE-induced T cell proliferation, the decreased number of CD3 and CD28 molecules as determined by FACS on CD11b- and CD11d-deficient T cells might explain at least part of the poor response of these cells to SE stimulation.
In comparison with WT splenocytes, the ratios of CD4+ to CD8+ cells in CD11b-KO and CD11d-KO splenocytes were markedly reduced. It is well known that CD4 and CD8 molecules contribute to the stable interaction of a complex of Ag-MHC molecules with the TCR molecule (42). Because superantigens activate T cells in direct association with MHC II molecule on APC (43, 44, 45, 46, 47, 48), it is expected that the toxins would preferentially stimulate CD4+ cells. Although some have suggested that SEs might affect CD4+ and CD8+ cells equally (7), others have found that CD4+ T cells were high responders to SEA, whereas CD8+ T cells were low responders in mice implanted with an SEA pump (42). Furthermore, an Ab to CD4 Ag blocked SEA-induced T cell response >80% (8). In addition, our study showed that CD4+ cells had higher intensities of CD3 expression than CD8+ cells in both WT and KO splenocytes, and the intensities of CD3 expression on both CD4+ and CD8+ cells were reduced in CD11b-KO and CD11d-KO mice compared with WT mice (data not shown). Based on these findings, the significantly lower ratios of CD4+to CD8+ cells in CD11b-KO and CD11d-KO splenocytes may be another reason for the reduced response of these cells to SE stimulation.
As an investigation in T cell development, cytometric analysis showed that the intensity of CD3 expression on CD11b- and CD11d-deficient thymocytes was also markedly reduced compared with that of WT thymocytes, and the ratios of CD4 to CD8 single-positive cells in CD11b-KO and CD11d-KO thymocytes were significantly lower than in WT thymocytes. In contrast, the ratios of CD4+ to CD8+ cells were normal in CD11a-KO thymocytes (27, 28). Our next expectation was that CD11b and CD11d might be expressed on WT mouse thymocytes and that the absence of CD11b or CD11d from the thymocytes as seen in CD11b-KO and CD11d-KO mice would result in changes in T cell phenotypes. Although expression of CD11b and CD11d was not detected on adult or 2-day-old mouse thymocytes, both CD11b and CD11d were expressed on early stage (day 12) WT mouse fetal thymocytes. In the early stage fetal thymocytes, the C-kit+ population has been previously described to consist of two populations (CD11b+ and CD11b– subsets) (49). Two major single-positive populations, Thy-1.2+IL-2R– and Thy-1.2–IL-2R+, appear to emerge from this C-kit+ population. These two major single-positive populations seem to be derived directly from the CD11b+ rather than from the CD11b– subset of the C-kit+ population. The fact that deficiency in either CD11b, in which case CD11d is present, or CD11d, in which case CD11b is present, results in similar T cell phenotypic changes indicates that both CD11b and CD11d are essential for early T cell development, and CD11b and CD11d must play distinct roles in this process. Because the majority of CD11d+ cells in mouse early fetal thymocytes are CD11b+, we postulate that this CD11b and CD11d double-positive population is critically important for early thymocyte differentiation. These results suggest that CD11b and CD11d transduce signals inwardly to thymocytes, and/or couple thymocytes to distinct cell partners. Absence of either CD11b or CD11d from the cells would affect early T cell development, resulting in the phenotypic changes in mature T cells shown in CD11b-KO and CD11d-KO mice, with reduced T cell responses to SE stimulation. In support of the role of CD11b and CD11d in early T cell development, we found that anti-CD11b and anti-CD11d Abs did not inhibit SEA-induced T cell proliferation in WT splenocytes in vitro (data not shown). Although previous reports have suggested that 1 integrins play a key role in thymocyte differentiation (50, 51), this is the first report that 2 (CD11/CD18) integrins also play an important role in thymocyte differentiation.
In summary, our study showed that T cells from CD11b-KO and CD11d-KO splenocytes had decreased proliferative response to SE stimulation, which may be related in part to phenotypic changes as evidenced by lower intensity of CD3 and CD28 expression on splenocytes, and lower ratios of CD4+ to CD8+ cells in the KO splenocytes. We also found a lower intensity of CD3 expression and lower ratio of CD4+ to CD8+ cells in the KO thymocytes. Both CD11b and CD11d were expressed on WT early stage fetal thymocytes. We postulate that both CD11b and CD11d are required for early stage thymocyte development, and absence of either CD11b or CD11d from the early stage fetal thymocytes leads to a defect in T cell development, resulting in reduced T cell response to SE stimulation.
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2 Address correspondence and reprint requests to Dr. Christie M. Ballantyne, Department of Medicine, Baylor College of Medicine, 6565 Fannin, Mail Stop A-601, Houston, TX 77030. E-mail address: cmb{at}bcm.tmc.edu
3 Abbreviations used in this paper: LAD I, leukocyte adhesion deficiency type I; SEA-SEE, staphylococcal enterotoxins A–E; KO, knockout; WT, wild type; MFI, mean fluorescence intensity.
Received for publication February 4, 2004. Accepted for publication April 26, 2004.
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