Regulatory role of CD8+ T lymphocytes in bone marrow eosinophilopoiesis

Background There is a growing body of evidence to suggest that CD8+ T lymphocytes contribute to local allergen-induced eosinophilic inflammation. Since bone marrow (BM) responses are intricately involved in the induction of airway eosinophilia, we hypothesized that CD8+ T lymphocytes, as well as CD4+ T lymphocytes, may be involved in this process. Methods Several approaches were utilized. Firstly, mice overexpressing interleukin-5 (IL-5) in CD3+ T lymphocytes (NJ.1638; CD3IL-5+ mice) were bred with gene knockout mice lacking either CD4+ T lymphocytes (CD4-/-) or CD8+ T lymphocytes (CD8-/-) to produce CD3IL-5+ knockout mice deficient in CD4+ T lymphocytes (CD3IL-5+/CD4-/-) and CD8+ T lymphocytes (CD3IL-5+/CD8-/-), respectively. Secondly, CD3+, CD4+ and CD8+ T lymphocytes from naïve CD3IL-5+ and C57BL/6 mice were adoptively transferred to immunodeficient SCID-bg mice to determine their effect on BM eosinophilia. Thirdly, CD3IL-5+, CD3IL-5+/CD8-/- and CD3IL-5+/CD4-/- mice were sensitized and allergen challenged. Bone marrow and blood samples were collected in all experiments. Results The number of BM eosinophils was significantly reduced in CD3IL-5+/CD8-/- mice compared to CD3IL-5+ mice and CD3IL-5+/CD4-/- mice. Serum IL-5 was significantly higher in CD3IL-5+/CD4-/- mice compared to CD3IL-5+ mice but there was no difference in serum IL-5 between CD3IL-5+/CD4-/- and CD3IL-5+/CD8-/- mice. Adoptive transfer of CD8+, but not CD4+ T lymphocytes from naïve CD3IL-5+ and C57BL/6 mice restored BM eosinophilia in immunodeficient SCID-bg mice. Additionally, allergen challenged CD3IL-5+/CD8-/- mice developed lower numbers of BM eosinophils compared to CD3IL-5+ mice and CD3IL-5+/CD4-/- mice. Conclusion This study shows that CD8+ T lymphocytes are intricately involved in the regulation of BM eosinophilopoiesis, both in non-sensitized as well as sensitized and allergen challenged mice.


Background
One important pathologic feature of allergic airway inflammation is associated with T lymphocyte activation and increase in eosinophil numbers in the airways [1][2][3]. Accumulation of eosinophils is considered to be the result of increased production and traffic of cells from the bone marrow (BM) into the airways via the circulation [4,5]. A substantial body of evidence suggests that BM eosinophilopoiesis is enhanced in allergic patients as well as in animal models of allergen-induced inflammation [6][7][8][9][10][11][12][13].
The allergen-induced increase in eosinophil numbers is closely linked to a Th 2 driven immune response based on the specific expression of cytokines exclusively secreted from CD4 + T lymphocytes [2,3]. In particular, the expression of interleukin-5 (IL-5) by T lymphocytes has been shown to be an essential signal necessary for the induction of eosinophilia in the airway [4,5,[14][15][16][17].
Whereas the pivotal role of CD4 + T helper (Th) cells in the development of allergic diseases has been demonstrated in several models, the exact role of CD8 + T lymphocytes remains unclear. Generally, the CD8 + T lymphocytes are considered to produce Th 1 cytokines, which is not always the case, since under certain circumstances CD8 + T lymphocytes also can produce Th 2 cytokines. For example, CD8 + T lymphocytes have been shown to produce IL-4, IL-5 and IL-13 following allergen stimulation [17][18][19][20].
An increasing amount of data suggests that CD8 + T lymphocytes contribute to allergen-induced airway inflammation. Depletion of CD8 + T lymphocytes prior to allergen challenge has been shown to decrease Th 2 cytokines, reduce eosinophil recruitment into the airway and reduce airway hyperresponsiveness [19][20][21][22]. Although CD8 + T lymphocytes appear to be involved in the regulation of local airway inflammation, less is known about their putative role in regulating distant pro-inflammatory responses, such as the enhanced eosinophilopoiesis seen after allergen exposure. We hypothesized that IL-5 producing CD8 + T lymphocytes may regulate BM responses following airway allergen exposure. To test this, we utilized an IL-5 transgenic mouse overexpressing IL-5 in CD3 + T lymphocytes (NJ.1638; CD3 IL-5+ ) that was bred with gene knockout mice lacking either CD4 + cells (CD4 -/ -) or CD8 + cells (CD8 -/-) in order to produce IL-5 transgenic-gene knockout mice deficient in CD4 + and CD8 + T lymphocytes, respectively. Bone marrow and blood samples were taken from offspring as well as from CD3 IL-5+ mice. Additionally, CD3 + , CD4 + or CD8 + T lymphocytes from naïve CD3 IL-5+ and wild type C57BL/6 mice were adoptively transferred to immunodeficient SCID-bg mice, in order to determine their role in regulating BM eosinophilia.

Methods
Mice IL-5 transgenic mice (NJ. 1638 (CD3 IL-5+ )) overexpressing IL-5 specifically in CD3 + T lymphocytes were obtained from Dr James J Lee (Mayo Clinic, Scottsdale, AZ, USA) and maintained in a heterozygous fashion by back-crossing to C57BL/6 mice. CD3 IL-5+ mice were bred with gene knockout mice lacking either CD4 + T lymphocytes (C57BL/6J CD4 tm1Knw ) or CD8 + T lymphocytes (C57BL/6 CD8a tm1Mak ) (Jackson Laboratories, Bar Harbor, ME) to produce CD3 IL-5+ knockout mice deficient in CD4 + and CD8 + T lymphocytes, respectively. Genotypes of mice produced by this crosses were assessed by the presence of CD3 IL-5+ and loss of T lymphocyte subtypes (PCR of tail DNA). Briefly, DNA was isolated from tail biopsies by using the DNeasy Tissue kit according to the manufacturer's instructions (Qiagen, Crawley, UK). The PCR reactions of DNA from C57BL/6 CD4 tm1Knw and C57BL/6 CD8a tm1Mak were prepared using the HotStartTaq Master Mix Kit (Qiagen, Crawley, UK) according to the protocol received from The Jackson Laboratory (Jackson Laboratories, Bar Harbor, ME). The PCR reactions of CD3 IL-5+ were assessed as previously described with some modifications [23].
Wild type C57BL/6 mice and C.B-17/Gbms Tac-SCID-bg mice were purchased from Mollegaard-Bommice A/S (Ry, Denmark). SCID-bg mice are immunodeficient mice that lack functional B and T-lymphocytes. All mice were provided with food and water ad libitum and housed in specific pathogen free animal facilities. The study was approved by the Ethics Committee for animal studies in Göteborg, Sweden.

Sample collection and processing
The animals were euthanized with a mixture of xylazin (130 mg/kg, Rompun ® , Bayer) and ketamine (670 mg/kg, Ketalar ® , Parke-Davis). First, blood was obtained by puncture of the heart right ventricle. Second, bronchoalveolar lavage (BAL) was performed by instilling 0.5 ml of phosphate buffered saline (PBS) through the tracheal cannula, followed by gentle aspiration and repeated with 0.5 ml PBS. Finally, bone marrow was harvested by excising one femur, which was cut at the epiphyses and flushed with 2 ml of PBS.

Blood
Two hundred microliters of blood was mixed with 800 μl of 2 mM EDTA (Sigma-Aldrich) in PBS, and red blood cells (RBC) were lysed in 0.1% potassium bicarbonate and 0.83% ammonium chloride for 15 min at RT. White blood cells (WBC) were resuspended in PBS containing 0.03% Bovine serum albumin (BSA, Sigma-Aldrich). For measurement of cytokines in serum the remaining volume of blood was centrifuged at 800 g for 15 min at 4°C.

Bone Marrow and Bronchoalveolar lavage fluid (BALF)
BM and BALF samples were centrifuged at 300 g for 10 min at 4°C. The cells were resuspended with 0.03% BSA in PBS. The total cell numbers in blood, BM and BALF were determined using standard hematological procedures. Cytospins of blood, bone marrow and BALF samples were prepared and stained according to the May-Grünwald-Giemsa method for differential cell counts. Cell differentiation was determined by counting 300-500 cells using a light microscope (Zeiss Axioplan 2, Carl Zeiss, Jena, Germany). The cells were identified using standard morphological criteria.

Sensitization and allergen exposure and in vivo labeling of newly produced eosinophils
Mice, 8-12 weeks old were sensitized on two occasions, five days apart by intraperitoneal (i.p) injections of 0.5 ml alum-precipitated antigen containing 8 μg Ovalbumin (OVA) (Sigma-Aldrich, St Louis, MO, USA) bound to 4 mg of Al(OH) 3 (Sigma-Aldrich) in PBS. Eight days after the second sensitization, the mice were rapidly and briefly anaesthetized with Isoflourane (Schering-Plough, UK), and received intranasal (i.n.) administration of 10 μg OVA in 25 μl PBS during five consecutive days. Twentyfour hours after the last OVA exposure the mice were sacrificed and cells from blood, BM and BALF were collected as described above. Additionally, the animals were given 5-Bromo-2'-deoxyuridine (BrdU) (Roche, Diagnostics Scandinavia AB, Bromma, Sweden) to label newly produced eosinophils. The BrdU was given at a dose of 1 mg in 250 μl PBS by i.p. injection twice, 8 hours apart on day 1 and on day 3 during OVA exposure.

Double immunostaining for nuclear BrdU and Major Basic Protein (MBP)
On day 1, cytospin preparations were fixed in 2% formaldehyde for 10 min and incubated with 10% rabbit serum (DAKO Corporation, Glostrup, Denmark) to avoid unspecific binding. BM and BALF slides were incubated with a monoclonal rat anti-mouse MBP antibody (kind gift from Dr James J Lee, Mayo Clinic, Scottsdale, AZ) for 1 hour followed by a 45 min incubation with alkaline phosphatase-conjugated rabbit F(ab') 2 anti-rat IgG secondary antibody (DAKO). Bound antibodies were visualized with Liquid Permanent Red substrate kit (DakoCytomation Inc, Carpenteria, CA, USA). Samples were fixed for a second time over night in 4% paraformaldehyde. On day 2, samples were treated with 0.1% trypsin (Sigma) at 37°C for 15 min followed by 4 M HCl for 15 min and Holmes Borate buffer (pH 8.5) for 10 min. Endogenous peroxidase was blocked with glucose oxidase solution (PBS supplemented with 0,0064% sodium azide, 0,18% glucose, 0,1% saponin and 1.55 units of glucose oxidase/ml PBS) preheated to 37°C for 30 min. BrdU labeled cells were detected using a FITC conjugated rat anti-mouse BrdU monoclonal antibody (clone BU1/75, Harlan-Sera Lab, Loughborough, UK), followed by a peroxidase conjugated rabbit anti-FITC secondary antibody (DAKO) and visualized with 3,3'-diaminobenzidine (DAB) substrate Chromogene System (DAKO). Mayer's Hematoxylin (Sigma) was used for counterstaining. Cells were determined by counting 400 cells using a light microscope (Zeiss Axioplan 2, Carl Zeiss, Jena, Germany).

Adoptive transfer experiments
Preliminary time-course experiments CD3 + lymphocytes from CD3 IL-5+ mice (10 7 cells in 0.35 ml 0.9% NaCl) or 0.9% NaCl alone was injected i.v to SCID-bg mice. Recipients were sacrificed on day 3, 10, 14, 21, 30 or 39 after cell transfer. Eosinophil numbers in BM and blood are shown in Table 1. In the final adoptive transfer experiments CD4 + , CD8 + or CD3 + lymphocytes (10 7 ) from CD3 IL-5+ or C57BL/6 mice in 0.35 ml of 0.9% NaCl or 0.9% NaCl alone was injected i.v to SCID-bg mice. All samples were obtained on day 39 after the transfer, which was based upon the most pronounced changes in BM and blood eosinophil numbers in the time-course experiment.

ELISA
Mouse IL-5 levels in serum were detected using commercial murine IL-5 ELISA kit (R&D Systems, Inc, Abingdon, UK). The lower detection limit was 3.9 pg/ml.

Statistical analysis
All data are expressed as mean ± SEM. Statistical analysis was carried out using a non-parametric analysis of vari-ance (Kruskal-Wallis test) to determine the variance among more than two groups. If significant variance was found, an unpaired two-group test (Mann-Whitney U test) was used to determine significant differences between individual groups. P < 0.05 was considered statistically significant.

Time-course experiment
A significant increase in blood eosinophils was evident on day 21 after transfer of CD3 cells from naïve CD3 IL-5+ to SCID-bg mice. A significant increase in BM eosinophils was not evident until 30 days after the cell transfer. The most pronounced increase in number of blood and BM eosinophils was observed 39 days after the cell transfer (Table 1). There were no time-dependent changes in BM eosinophils in the 0.9% NaCl-injected control groups.

Serum IL-5 in SCID-bg mice after adoptive transfer of CD3 IL-5+ CD3 + , CD4 + or CD8 + T cells
Transfer of CD3 IL-5+ CD3 + , CD4 + and CD8 + splenocytes induced a substantial increase in the concentration of recipient serum IL-5. There were no significant differences in the concentration of serum IL-5 between transfer groups (Fig. 2C).

Blood
There was no difference in blood eosinophilia in any of the transferred groups compared to the 0.9% NaClinjected control mice.
Recent data is suggesting that not only CD4 + T lymphocytes, but also CD8 + T lymphocytes, contribute to allergen-induced airway inflammation. Depletion of CD8 + T lymphocytes prior to allergen challenge has been shown to decrease Th 2 cytokines, reduce eosinophil recruitment into the airway and reduce airway hyperresponsiveness [19][20][21][22]. Although CD4 + and CD8 + T lymphocytes appear to be involved in the regulation of local airway inflammation, less is known about their role in BM eosinophilopoiesis after allergen exposure. The number of CD3 + T lymphocytes expressing IL-5 mRNA and protein is increased in BM, circulation as well as in the airways following allergen challenge in both mice and humans [5,[15][16][17]. Therefore, in the present study we utilized IL-5 transgenic mice (CD3 IL-5+ ) that constitutively overexpress Eosinophil numbers after adoptive transfer of C57BL/6 CD3 + , CD4 + or CD8 + T cells to SCID-bg mice Figure 3 Eosinophil numbers after adoptive transfer of C57BL/6 CD3 + , CD4 + or CD8 + T cells to SCID-bg mice. Eosinophils in BM of naïve SCID-bg mice 39 days after adoptive transfer of CD4 + , CD8 + and CD3 + T cells enriched from naïve C57BL/6 mice. Data are shown as mean (+SEM) (n = 6-7). *P < 0.05 increased from control treated mice. IL-5 in CD3 + T lymphocytes [23], which is known to result in an enhanced eosinophilopoiesis and increased levels of circulating eosinophils [7,23]. Importantly, we have recently shown that adoptive transfer of CD3 + T lymphocytes from sensitized CD3 IL-5+ mice induced an increase in BM eosinophils in allergen-exposed recipient wild type mice [7].
To assess the role of CD4 + and CD8 + T lymphocytes in BM eosinophilopoiesis we crossbred gene knockout mice deficient in CD4 + or CD8 + T lymphocytes with CD3 IL-5+ mice. Notably, CD3 IL-5+ mice deficient in CD8 + T lymphocytes had a reduced number of BM eosinophils compared to CD3 IL-5+ mice or CD3 IL-5+ deficient in CD4 + T lymphocytes. Initially, we hypothesized that this could be due a difference in IL-5 production between the crossbred mice, since CD8 + T lymphocytes can produce several Th 2 cytokines including IL-5 [19,20]. A significant increase in serum IL-5 levels was found in CD3 IL-5+ mice deficient in CD4 + T lymphocytes compared to CD3 IL-5+ mice. It could be speculated that this phenomena is due to a lack of T regulatory cells in these mice. However, we were not able to find any difference in serum IL-5 between the two crossbred strains, indicating that CD8 + T lymphocytes are required to maintain high levels of a strongly IL-5 dependent BM eosinophilopoiesis. Importantly, our present study further shows that adoptive transfer of CD3 IL-5+ CD8 + T lymphocytes as well as transfer of CD8 + T lymphocytes from C57BL/6 mice restored BM eosinophilia in immunodeficient (SCID-bg) mice. The finding that not only transfer of CD3 IL-5+ CD8 + T lymphocytes but also transfer of CD8 + T lymphocytes from C57BL/6 mice restore BM eosinophilia in immunodeficient mice further argues that the role of CD8 + T lymphocytes in BM eosinophilopoiesis is independent of IL-5 overproduction. Importantly, IL-5 is not only produced by CD4 + T lymphocytes, but also CD8 + T lymphocytes, as well as CD34 + cells. The initial development of eosinophilia is induced in a complex way, including T lymphocyte independent mechanisms, as well as production of IL-5 from CD34 + cells [14,24]. CD8 + T lymphocytes probably interact in this process both by IL-5 dependent as well as IL-5 independent mechanisms (Figure 2A and 3, respectively).
In allergen-exposure experiments, we further show that CD8 + T lymphocytes are involved also in allergen-induced BM eosinophilopoiesis. In this experiment, we stained cells with a monoclonal antibody to eosinophil granule major basic protein (MBP), since is known that this is expressed early on eosinophil-committed cells [25,26]. Allergen exposed CD3 IL-5+ /CD8 -/mice showed a reduction of BM MBP + eosinophils compared to CD3 IL-5+ mice, whereas in the CD3 IL-5+ /CD4 -/mice the number of BM MBP + eosinophils remained unchanged compared to CD3 IL-5+ mice. One explanation to this could be a reduced production of eosinophils in the CD3 IL-5+ /CD8 -/mice. We directly addressed this question by using a double staining technique for newly produced eosinophils (i.e. BrdU + /MBP + cells). However, we where not able to show any significant reduction in BrdU + /MBP + BM eosinophils in any of the crossbred strains compared to CD3 IL-5+ mice, although the CD3 IL-5+ /CD8 -/mice showed a trend of a reduction in BrdU + /MBP + eosinophils. It could be speculated that the production of eosinophils in the BM has a rapid turnover in these mice and that the newly produced cells are released in to the circulation and already accumulated in the airways.
Notably, when CD4 + T lymphocytes were eliminated, almost no recruitment of eosinophils into the airways occurred. However, for the restoration of the allergeninduced eosinophil recruitment into the airways, both CD4 + and CD8 + T lymphocyte subsets may be required, which is in agreement with a recent report [20]. It has been previously shown that CD4 + T lymphocytes are required for traffic of eosinophils to airways, also in mice that excessively overexpress IL-5 in the airway epithelium [27]. Thus, CD4 + T lymphocytes are contributing to eosinophil traffic to airways in parallel to IL-5. However, our present study also shows that when CD8 + T lymphocytes are lacking in a mouse overexpressing IL-5 in CD3 + T lymphocytes, a reduction in the recruitment of eosinophils to the airways occur. This seems to be a reflection of a reduced production of eosinophils in the BM in CD8 + T lymphocyte deficient mice. Furthermore, it has recently been shown that CD8 + T lymphocytes are a source of IL-13 [22]. Therefore depletion of CD8 + T lymphocytes may partly reduce airway eosinophilia as a consequence of a reduction in IL-13, since it has been reported that administration of IL-13, or overexpression of IL-13 in the airways, induces eosinophilia [28,29].

Conclusion
In summary, we here show for the first time that CD8 + T lymphocytes regulate BM eosinophilopoiesis both at baseline and after allergen exposure. In the presence of IL-5, CD8 + T lymphocytes seem to be required for the maintenance of eosinophil production in the BM, while CD4 + T lymphocytes are required for their recruitment into the airways following airway allergen exposure. Thus, CD8 + T lymphocytes are involved in some of the systemic processes in allergic eosinophilia, which has implications in understanding the overall complex mechanisms of allergic diseases.