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The Journal of Immunology, 1999, 162: 5957-5964.
Copyright © 1999 by The American Association of Immunologists

Expansion of Extrathymic T Cells as Well as Granulocytes in the Liver and Other Organs of Granulocyte-Colony Stimulating Factor Transgenic Mice: Why They Lost the Ability of Hybrid Resistance1

Hiroki Kawamura*, Toshihiko Kawamura*, Yasuo Kokai{dagger}, Michio Mori{dagger}, Akihiro Matsuura{dagger}, Hiroshi Oya*, Shigeru Honda*, Susumu Suzuki*, Anura Weerashinghe*, Hisami Watanabe* and Toru Abo2,*

* Department of Immunology, Niigata University School of Medicine, Niigata, Japan; and {dagger} Department of Pathology, Sapporo Medical University, Sapporo, Japan


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
When we attempted to characterize the immunological state in G-CSF transgenic mice, a large number of not only granulocytes but also lymphoid cells expanded in various immune organs. Such lymphoid cells were present at unusual sites of these organs, e.g., the parenchymal space in the liver. We then determined the phenotype of these lymphoid cells by immunofluorescence tests. It was demonstrated that CD3intIL-2Rß+ cells (i.e., extrathymic T cells), including the NK1.1+ subset of CD3int cells (i.e., NKT cells), increased in the liver and all other tested organs. These T cells as well as NK cells mediated NK and NK-like cytotoxicity, especially at youth. However, they were not able to mediate such cytotoxicity in the presence of granulocytes. This result might be associated with deficiency in the hybrid resistance previously ascribed to these mice. In other words, G-CSF transgenic mice had a large number of extrathymic T cells (including NKT cells) and NK cells that mediate hybrid resistance, but their function was suppressed by activated granulocytes. Indeed, these granulocytes showed an elevated level of Ca2+ influx upon stimulation. The present results suggest that, in parallel with overactivation of granulocytes, extrathymic T cells and NK cells are concomitantly activated in number but that their function is suppressed in G-CSF transgenic mice.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Mice transgenic for human G-CSF (called G-Tg mice hereafter)3 were recently established by one group of the authors of this paper (1). Human G-CSF is effective in mice. Reflecting this situation, these G-Tg mice showed an extraordinary expansion of granulocytes in the bone marrow and in other peripheral immune organs. However, hemopoietic stem cells and lymphoid cells were also found to increase in number in the bone marrow and periphery of these mice (2). Although these lymphoid cells did not seem to be conventional T and B cells, further phenotypic and functional characterization remained to be investigated.

In parallel with this numerical change of leukocytes, these G-Tg mice were found to lose the ability of hybrid resistance (3, 4), the meaning of which is as follows. When mice are lethally irradiated (e.g., 9.5 Gy) and subjected to semiallogeneic or allogeneic bone marrow cells, they are still able to reject such injected semiallogeneic or allogeneic cells (i.e., natural resistance against allogeneic bone marrow cells) (5, 6, 7, 8). Radioresistant NKT cells and NK cells are responsible for this hybrid resistance phenomenon (9, 10, 11). The numerical and functional properties of these NKT cells in G-Tg mice also remained to be examined.

In this study, we first examined the histology of the liver and various immune organs in G-Tg mice and observed an interesting phenomenon. Namely, lymphoid cells that increased in the liver of these mice localized at unusual sites such as the parenchymal space of the liver. In a series of recent studies, we characterized the phenotype (12, 13, 14, 15, 16), function (17, 18, 19), and the differentiation pathway (20, 21, 22, 23, 24, 25, 26) of extrathymic T cells that exist at various sites in mice. The above-mentioned site where lymphoid cells are present in G-Tg mice resembles those for extrathymic T cells in normal mice. Thus, conventional T cells (i.e., thymus-derived T cells) localize in the lymph nodes, the white pulp of the spleen, and the sinusoidal lumen in the liver, whereas extrathymic T cells exist in the parenchymal space of the liver (27, 28). In light of these findings, in the present study, we investigated the possibility that lymphoid cells expanding in G-Tg mice are extrathymic T cells rather than conventional T cells. The present data indicate that they belonged to extrathymic T cells. We also discussed why such extrathymic T cells expanded in number in parallel with granulocytes in these G-Tg mice.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice

Wild C57BL/6 (B6) and G-Tg (B6 background) mice were maintained at the animal facility of Niigata University. The establishment of these G-Tg mice has been previously described (1, 2). These G-Tg mice were back-crossed for more than eight generation. All mice were fed under specific pathogen-free conditions.

Restraint stress and indomethacin treatment

G-Tg mice showed a susceptibility to stress, especially when they were exposed to restraint stress or were administered with a nonsteroidal antiinflammatory drug (NSAID), indomethacin. They easily fall victim to hepatic or renal failure. Mice were subjected to restraint stress for 24 h, whereas indomethacin (15 mg/mouse) was orally administered. Immediately after the stress, or 3 days after the administration of indomethacin, mice were sacrificed to examine immunoparameters.

Cell preparations

Mice were anesthetized with ether and sacrificed by total bleeding from the incised axillary artery and vein. The organs to be used for the experiments were removed, and mononuclear cells (MNC) were obtained as follows. Hepatic MNC were isolated by a previously described method (27). Briefly, the liver was cut into small pieces with scissors, pressed through 200-gauge stainless steel mesh, and suspended in Eagle’s MEM medium (Nissui Pharmaceutical, Tokyo, Japan) supplemented with 5 mM HEPES (Sigma, St. Louis, MO) and 2% heat-inactivated newborn calf serum. After being washed once with the medium, MNC were isolated from both hepatocytes and the nuclei of hepatocytes by the Percoll (35% Percoll containing 100 U/ml heparin) gradient method (27). Spleen cells, thymocytes, and lymph node cells were obtained by forcing the spleen, thymus, and inguinal lymph nodes through 200-gauge steel mesh. Bone marrow cells were obtained by flushing femurs with PBS. Blood MNC were isolated by Ficoll-Isopaque gradient (1.094) centrifugation. Splenocytes and bone marrow cells were used after erythrocyte lysing. Erythrocyte lysing solution consisted of 155 mM NH4Cl, 10 mM KHCO3, 1 mM EDTA-Na, and 170 mM Tris, pH 7.3.

Flow cytometric analysis

The surface phenotype of cells was analyzed using mAbs in conjunction with either a two- or a three-color immunofluorescence test (29). The mAbs used here included FITC-, PE-, or biotin-conjugated reagents of anti-CD3 (145-2C11), anti-IL-2Rß (TM-ß1), anti-NK1.1 (PK136), anti-CD4 (RM4-5), anti-CD8 (53-6.7), anti-Mac-1 (M1/70), and anti-Gr-1 (RB6-8C5) mAbs (PharMingen, San Diego, CA). Biotin-conjugated reagents were developed with either PE- or Red 613-conjugated streptavidin (Becton Dickinson, Mountain View, CA). To prevent nonspecific binding of mAbs, CD32-16 (2.4G2) was added before staining with labeled mAbs (29). The fluorescence-positive cells were analyzed by FACScan (Becton Dickinson). Dead cells were excluded according to their forward scatter, side scatter, and propidium iodide (PI) gating. Hepatic lymphocytes depleted of granulocytes (Gr-1- cells) in G-Tg mice were also prepared using a cell sorter, FACStar II Plus (Becton Dickinson).

Cytotoxicity assay

Target cells were NK-sensitive YAC-1 cells. Cytotoxic activity was measured by a specific 51Cr-release assay (18). Labeled targets (104/well) were incubated in a total volume of 200 µl with effector cells in RPMI 1640 medium supplemented with 10% FCS in a 96-well round-bottomed microculture plate. Incubation was performed for 4 h. Effector cells were liver leukocytes or liver lymphocytes depleted of granulocytes.

Measurement of cytosolic Ca2+ in granulocytes

MNC isolated from the liver, spleen, blood, and bone marrow (2 x 106 cells/tube) were incubated for 30 min at 37°C in the dark with Fluo-3/AM (Molecular Probes, Eugene, OR) at a concentration of 1 µM. The concentrations of cytosolic free Ca2+ were measured using Fluo-3 fluorescence (30). Fluo-3-loaded cells were excited with an argon laser at 488 nm, and fluorescence was measured at 525 nm. The Fluo-3 fluorescence of unstimulated cells (basal fluorescence) was set to an arbitrary unit. Cells were preincubated at 24°C in the dark for 10 min. After measurement of basal fluorescence, cells were stimulated by the addition of fMLP (10-5 M).

Measurement of plasma concentration of noradrenaline

Plasma pooled from four mice was used to measure the concentration of adrenaline, noradrenaline, dopamine, and corticosterone. The plasma levels of these catecholamines were analyzed by the HPLC method (30).

Statistical analysis

Statistical significance was analyzed by Student’s t test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Expansion of lymphoid cells as well as granulocytes in the liver of G-Tg mice

It was previously reported that G-Tg mice had an extraordinally high number of granulocytes in various tissues (1, 2). However, we observed that lymphoid cells other than granulocytes were also found in high number in the liver of these mice (Fig. 1Go); for example, a massive accumulation of lymphoid cells, as well as granulocytes, was seen in the parenchymal space. The ratio of granulocyte:lymphocyte in the liver of G-Tg mice was determined later by immunofluorescence tests.



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  		FIGURE 1. A comparison of histology of the liver between control B6 mice and G-Tg mice (magnification, x400). a, The liver of control B6 mice. b, The liver of G-Tg mice. Regular hematoxylin-eosin staining was conducted.

 
Generalized expansion of IL-2Rß+CD3int cells as well as granulocytes in G-Tg mice

To investigate what kinds of lymphoid cells expanded in G-Tg mice, leukocytes were isolated from various immune organs. The cell yields by these organs were first enumerated (Fig. 2Go). The data from age-matched wild B6 mice are represented in parallel. Although the number of leukocytes yielded by the thymus was comparable, those yielded by all other tested organs increased (p < 0.05). The increase in the number of leukocytes in the liver, spleen, and peripheral blood was especially remarkable (p < 0.001). As suggested from this data, the splenomegaly seen in G-Tg mice was extraordinal.



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  		FIGURE 2. Increase in the number of cells yielded by the liver, spleen, and peripheral blood in G-Tg mice. Control and G-Tg mice at the age of 10 wk were examined. The mean and one SD were produced by five mice of each group.

 
Phenotypic characterization of leukocytes was conducted by two-color staining with mAbs in various combinations (Fig. 3Go). Two-color staining for CD3 and IL-2Rß was first conducted to identify NK cells (CD3-IL-2Rß+), extrathymic T cells (CD3intIL-2Rß+), and conventional T cells (CD3highIL-2Rß-) (Fig. 3Go, left column). In all tested organs, the proportion of CD3intIL-2Rß+ cells was found to increase prominently in G-Tg mice. The proportion of CD3-IL-2Rß+ NK cells also increased in the liver and peripheral blood of these mice. Because CD3intIL-2Rß+ cells are known to comprise NKT cells (i.e., CD3intNK1.1+ cells) (16), two-color staining for CD3 and NK1.1 was then conducted (Fig. 3Go, second column). CD3intNK1.1+ cells (i.e., NKT cells) were present in the liver of control mice and in the liver, thymus, bone marrow, and peripheral blood of G-Tg mice. Concerning the proportion of NKT cells in G-Tg mice, 50% of the CD3int cells in these mice were NKT cells in the liver, thymus, and bone marrow, whereas <50% of the CD3int cells were NKT cells in the spleen and peripheral blood.



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  		FIGURE 3. Phenotypic characterization of cells in various immune organs of control and G-Tg mice. Control and G-Tg mice at the age of 10 wk were examined. Two-color staining for CD3 and IL-2Rß, CD3 and NK1.1, CD4 and CD8, and Mac-1 and Gr-1 were conducted. Numbers represent the percentages of fluorescence-positive cells in corresponding areas. The data shown are representative of three experiments.

 
Two-color staining for CD4 and CD8 was conducted to determine the distribution of CD4+ and CD8+ cells (Fig. 3Go, third column). No great difference in distribution was observed between the control (wild) mice and the G-Tg mice.

Usually, the level of granulocytes is very low in various immune organs of normal mice (30). They are abundant only in the bone marrow. However, when leukocytes in various organs of G-Tg mice were examined by the cell analyzer, there were cells with unique light scatter similar to that of granulocytes. In this regard, two-color staining for Mac-1 and Gr-1 was conducted to determine granulocytes (Gr-1+Mac-1+) and macrophages (Gr-1-Mac-1+) (Fig. 3Go, right column). It was found that either granulocytes or macrophages increased proportionally in the liver, spleen, bone marrow, and peripheral blood of G-Tg mice.

Elevated level of NK and NK-like cytotoxicity in the liver of G-Tg mice

We previously reported that NK and NKT cells, which are abundant in the liver, mediate potent NK and NK-like cytotoxicity, respectively (17, 18). Because the proportion of NK and NKT cells increased in the liver of G-Tg mice, we then examined whether they could mediate such NK and NK-like cytotoxicity (Fig. 4GoA). As expected, liver leukocytes isolated from control mice showed a high level of cytotoxicity. However, it was found that such cytotoxicity was very low in G-Tg mice. Liver leukocytes (20% granulocytes) isolated from G-Tg mice were then depleted of granulocytes (Gr-1- cells). It was demonstrated that Gr-1- cells (<1% granulocytes) that contained NK cells and NKT cells mediated greater NK and NK-like cytotoxicity than did liver leukocytes isolated from control mice. A similar tendency resulted with age (50 wk) in both control and G-Tg mice.



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  		FIGURE 4. A comparison of NK and NK-like cytotoxicity in the liver between wild and G-Tg mice. a, Depletion of granulocytes from liver leukocytes in G-Tg mice. b, Addition of granulocytes isolated from G-Tg mice into liver MNC of wild mice. Wild and G-Tg mice were used at the ages of 10 and 50 wk. To determine NK-like cytotoxicity, YAC-1 targets (104/well) were used with effector cells at the indicated E:T ratios. Because liver leukocytes contained a large number of granulocytes (20%) in G-Tg mice, both whole liver leukocytes and liver leukocytes depleted of granulocytes by a cell sorter (Gr-1- cells) were examined as effector cells. To show the suppressive effect of granulocytes on the cytotoxicity, granulocytes isolated from the bone marrow of G-Tg mice were added into liver MNC in wild mice at the indicated proportion. Here, the number of liver MNC remained constant. The mean and one SD of triplicate cultures are represented.

 
We further conducted the cell-addition experiment to show that activated granulocytes eventually mediated the suppressive effect on NK and NK-like cytotoxicity (Fig. 4GoB). In this experiment, we added granulocytes of G-Tg mice to liver leukocytes of wild mice. Granulocytes were isolated from the bone marrow of G-Tg mice (the purity was 80%). A significant suppressive effect was reproduced, although the elimination experiment of granulocytes was much more prominent in terms of the magnitude. This might be due to the isolated procedure for granulocytes from G-Tg mice. Their function decreased slightly because living granulocytes were fragile in vitro.

Age-associated change of lymphocyte subsets in the liver and spleen of G-Tg mice

The above experiments showed that not only the proportion of granulocytes but also those of NK and extrathymic T cells increased in young G-Tg mice. Therefore, it was investigated whether such changes were consistent even in older G-Tg mice (Fig. 5Go). In these experiments, two-color staining for CD3 and IL-2Rß and that for CD3 and NK1.1 were conducted in G-Tg mice at various ages. Control stainings of normal mice at the age of 20 wk are represented in parallel. In both the liver and spleen of G-Tg mice, the proportion of NK cells and extrathymic T cells tended to increase from 10 to 30 wk of age. However, these NK cells as well as NKT cells (i.e., NK1.1+ subset of CD3int cells) tended to decrease both in the liver and spleen at older ages. In other words, the NK1.1- subset of extrathymic T cells did not decrease even at older ages. As previously shown, NK cells increase in number as a function of age and finally the number declines (31). Therefore, we estimate that G-Tg mice show an accelerated aging.



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  		FIGURE 5. Age-associated change in the phenotype of cells in various immune organs of G-Tg mice. G-Tg mice at the indicated ages and wild mice at the age of 20 wk were used. The phenotype of cells in the liver and spleen was examined. In this experiment, two-color staining for CD3 and IL-2Rß and that for CD3 and NK1.1 were conducted. Numbers represent the percentages of fluorescence-positive cells in corresponding areas.

 
Age-associated increase in the proportion of granulocytes in G-Tg mice

Two-color staining for Gr-1 and Mac-1 was conducted in various immune organs to determine the age-associated increase of granulocytes in G-Tg mice. The actual proportion of Gr-1+Mac-1+ granulocytes are plotted (Fig. 6Go). In both the bone marrow and peripheral blood of G-Tg mice, an age-associated increase in the proportion of granulocytes was observed.



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  		FIGURE 6. Age-associated change in the proportion of granulocytes in various immune organs of G-Tg mice. The percentage of granulocytes was determined by two-color staining for Mac-1 and Gr-1. The percentage of granulocytes (Mac-1+Gr-1+) was produced from four mice at each point of time. The data shown here are representative of three experiment.

 
Functional activation of granulocytes isolated from various organs of G-Tg mice

Granulocytes have the ability to produce superoxides, including H2O2 and myeloperoxidase (30). These activities are highly associated with the magnitude of initial Ca2+ influx after stimulation with fMLP. It was then compared whether granulocytes isolated from control B6 mice and those from G-Tg mice showed any difference in such Ca2+ influx (Fig. 7Go). Granulocytes were isolated from the liver, spleen, peripheral blood, and bone marrow of each group of mice. It was clearly demonstrated that granulocytes isolated from G-Tg mice had a higher ability of Ca2+ influx than did those from control mice.



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  		FIGURE 7. A comparison of Ca2+ influx by granulocytes between wild B6 mice and G-Tg mice. Ca2+ influx was measured by an immunofluorescence test, before and after the stimulation by fMLP. Granulocytes were obtained from the bone marrow of mice of each group.

 
Increase in the production of adrenaline but decrease in the production of corticosterone in G-Tg mice

We previously reported that there are two types of regulation of granulocytes in number and function: 1) sympathetic nerve stimulation increases the number and function of granulocytes that bear surface adrenergic receptors, and 2) superoxide production of granulocytes induces sympathetic nerve activation (32, 33, 34). In this experiment, we examined whether the serum level of catecholamines was elevated in G-Tg mice (Fig. 8Go). The serum level of corticosterone was examined in parallel. Interestingly, there was a dissociation among the levels of catecholamines, because the level of adrenaline increased while those of noradrenaline and dopamine decreased (p < 0.01). The profoundly decreased serum level of corticosterone in G-Tg mice was also noteworthy. These results are in agreement with the appearance of the adrenal gland in G-Tg mice, showing the atrophic cortex and hyperplasic medulla (data not shown). As a result, G-Tg mice looked unhealthy.



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  		FIGURE 8. Serum level of catecholamines and that of corticosterone in wild mice and G-Tg mice. Serum was isolated from mice of each group, and adrenaline, noradrenaline, dopamine, and corticosterone were measured. The mean and one SD were produced from three mice.

 
A comparison of the response to restraint stress and indomethacin treatment between wild mice and G-Tg mice

Restraint stress and NSAID treatment are known to simultaneously activate granulocyte and extrathymic T cells in number and function (30, 34). As a result, such activated granulocytes and extrathymic T cells invade mucosal tissues and organs such as the stomach and liver and induce gastric ulcers and hepatic failure, respectively. In this experiment, we conducted such restraint stress and NSAID treatment in wild mice and G-Tg mice (Fig. 9Go). Similar to the case of wild mice, the activation of extrathymic T cells (i.e., CD3intIL-2Rß+) and granulocytes (i.e., Mac-1+Gr-1+) was induced by both restraint stress and indomethacin treatment. These mice showed elevated levels of transaminases, suggesting hepatic failure (data not shown). In almost all of the organs, the accumulation of granulocytes and extrathymic T cells was much more prominent in G-Tg mice than in wild mice. These results explain the unhealthy state and the lability to stress seen in G-Tg mice.



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  		FIGURE 9. Variation of lymphocyte subsets and myeloid cells when G-Tg mice were exposed to restraint stress and were orally administered with indomethacin. Wild mice and G-Tg mice at the age of 10 wk were used. Mice were exposed to restraint stress for 24 h or orally administered with indomethacin (15 mg/mouse). Leukocytes were obtained from various immune organs just after the stress or 24 h after the administration of indomethacin. Numbers in the represent percentages of fluorescence-positive cells in corresponding areas. The data shown are representative of three experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present study, we further characterized G-Tg mice for three purposes: 1) determination of the phenotype of lymphoid cells, which expanded in parallel with granulocytes, 2) why these mice lost the ability to eliminate allogeneic bone marrow cells (i.e, hybrid resistance), and 3) the reason for susceptibility to stress in these mice. Usually, only a few lymphocytes are present in the parenchymal space of the liver (27, 28). However, these G-Tg mice showed an abundance of lymphoid cells in the parenchymal space of the liver. The numbers of cells yielded by the liver, spleen, and blood in G-Tg mice were higher than those in control mice. Interestingly, the expanding lymphocyte subsets in these mice were estimated to be extrathymic T cells and NK cells. In control wild mice, extrathymic T cells and NK cells were abundant only in the liver. However, in the case of G-Tg mice, extrathymic T cells and NK cells increased in proportion and in absolute number in all tested organs. Primarily, extrathymic T cells showed an inverted expression of adhesion molecules (e.g., CD44+L-selectin-) from that of conventional T cells (e.g., CD44-L-selectin+) (15, 35, 36). This was true in extrathymic T cells in the liver of G-Tg mice (our unpublished observation). Therefore, it is speculated that the expanding lymphoid cells in G-Tg mice were present at unique sites such as the parenchymal space of the liver.

We previously reported that extrathymic T cells (i.e., CD3int cells) comprise NK1.1+ and NK1.1- subsets at a ratio of 1:1 (16). The NK1.1+ subset of CD3int cells have also been called NKT cells in recent studies (37, 38, 39, 40, 41, 42). Despite the extraordinary expansion of CD3int cells in various organs of G-Tg mice, the ratio of NK1.1+ and NK1.1- subsets did not significantly vary. The NK1.1+ subset of CD3int cells was extremely radioresistant, namely, they remained in the bone marrow even after lethal irradiation (e.g., 9.5 Gy) (43). Primarily, they have the potential to recognize alloantigens (i.e., allogeneic polymorphic MHC) (43), as well as to recognize self-Ags in the context of monomorphic MHC Ags such as CD1 and TL Ags (44, 45, 46). In this regard, these NKT cells act as effector cells to reject allogeneic bone marrow cells (i.e., hybrid resistance) when these bone marrow cells are injected into lethally irradiated allogeneic mice. Despite the extraordinary expansion of NKT cells, it was strange that these G-Tg mice lost the ability of hybrid resistance. The results from the functional assay of liver lymphocytes in these mice taught us the reason. Thus, although purified leukocytes mediated NK-like cytotoxicity, their mixture with granulocytes could not mediate such cytotoxicity. It is speculated that activated granulocytes in G-Tg mice suppressed the function of NKT cells and NK cells. The elimination experiments and cell-addition experiments support this notion. In the case of old G-Tg mice, even purified lymphocytes from granulocytes showed a slightly decreased level of cytotoxicity.

It is widely known that granulocytes can produce superoxides (and other reactive oxygen intermediates) under stimulation by various bacterial components, stressors, and even NSAIDs (30, 34, 47, 48). In this regard, overactivation of granulocytes sometimes induces tissue damage. In contrast, extrathymic T cells do not completely eliminate self-reactive forbidden clones during their differentiation and maturation (17, 18, 19, 20, 21). As a result, they are able to mediate self-reactive cytotoxicity against rapidly proliferating self-cells and malignant tumors (24, 25). Under usual conditions, this function seems to be beneficial. However, similar to the case of granulocytes, it is presumed that overactivation of extrathymic T cells might be responsible for the susceptibility to diseases. Indeed, these G-Tg mice are susceptible to tissue damage when exposed to stress or NSAIDs (our unpublished observation).

CD3int cells, especially the NK1.1+ subset (i.e., NKT cells), were found to be generated extrathymically in the liver from preexisting c-kit+ stem cells (23, 26). One of the main growth factors for CD3int cells is IL-7, which is produced by parenchymal hepatocytes (22). However, we also demonstrated that CD3int cells express G-CSF receptors that were detected by the RT-RCR method (49). At that time, we did not know why these G-CSF receptors were present on CD3int cells. As shown previously, we reported the simultaneous activation of granulocytes and extrathymic T cells in number and function when mice were exposed to restraint stress and were administered with NSAID (30, 34). In conjunction with the data on the simultaneous expansion of granulocytes and extrathymic T cells in G-Tg mice, these two leukocyte populations seem to be regulated in a similar fashion, possibly via their surface G-CSF receptors. We have recently begun to call extrathymic T cells primordial T cells, because T cells with properties similar to those of extrathymic T cells are generated through an alternative pathway of T cell differentiation in the thymus (20). Therefore, we conclude that these primordial T cells still have a regulatory mechanism similar to that of myeloid cells.

Even in humans, some patients with a high level of granulocytes in the peripheral blood always show a high level of extrathymic T cells (i.e., human extrathymic T cells, including CD56+ T cells and CD57+ T cells). Such diseases include rheumatoid arthritis, malignancy, AIDS, etc. (50, 51). These patients show a compromised function of immune cytotoxicity, i.e., profoundly suppressed levels of NK-like cytotoxicity (our unpublished observations). It is speculated that activated granulocytes suppress such immune cytotoxicity in these patients, resulting in susceptibility to infectious diseases.


   Acknowledgments
 
We thank Mrs. Masako Watanabe for manuscript preparation and Mr. Tetsuo Hashimoto for animal maintenance.


   Footnotes
 
1 This work was supported by a grant-in-aid for scientific research from the Ministry of Education, Science, and Culture, Japan. Back

2 Address correspondence and reprint requests to Dr. Toru Abo, Department of Immunology, Niigata University School of Medicine, Asahimachi-1, Niigata 951-8510, Japan. E-mail address: Back

3 Abbreviations used in this paper: G-Tg mice, G-CSF transgenic mice; IL-2Rß, IL-2R ß-chain; CD3int cells, intermediate CD3 cells; MNC, mononuclear cells; B6, C57BL/6; PI, propidium iodide; NKT, NK1.1+ subset of CD3int cells; NSAID, nonsteroidal antiinflammatory drug. Back

Received for publication October 13, 1998. Accepted for publication March 1, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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