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Lipocalin 2 functions as a negative regulator of red blood cell production in an autocrine fashion

Ken-ichi Miharada^*,, Takashi Hiroyama^*, Kazuhiro Sudo^*, Toshiro Nagasawa and Yukio Nakamura^*,1

^* Cell Engineering Division, BioResource Center, RIKEN, Tsukuba, Ibaraki, Japan; and
Division of Hematology, Institute of Clinical Medicine, University of Tsukuba, Tsukuba, Ibaraki, Japan

¹ Correspondence: Cell Engineering Division, BioResource Center, RIKEN, 3-1-1 Koyadai, Tsukuba, Ibaraki 305-0074, Japan. E-mail: yukionak{at}brc.riken.jp

SPECIFIC AIMS

Members of the lipocalin protein family are typically small, secreted proteins that possess a variety of functions, including the regulation of the immune response and the mediation of cell homeostasis. Although the physiological role of lipocalin 2 remains to be fully elucidated, a few pivotal functions have recently been reported. These functions include regulation of the apoptosis of leukocytes, the transport of iron, and inhibition of bacterial growth by sequestration of the iron-laden siderophore. Initially, we unexpectedly found that lipocalin 2 is abundantly expressed in erythroid progenitor cells, progenitors of red blood cells (RBCs). To delineate the roles of lipocalin 2 in erythropoiesis, we assessed the functions of lipocalin 2 in the in vitro and in vivo models of erythropoiesis.

PRINCIPAL FINDINGS

1. Erythroid progenitor cells express and secrete lipocalin 2
Human lipocalin 2 has been described as the neutrophil gelatinase-associated lipocalin (NGAL). The expression of lipocalin 2 has also been detected in nonhematopoietic tissues, such as epithelia and the uterus, prompting us to analyze the expression of lipocalin 2 in various hematopoietic lineages. The expression of lipocalin 2 was detected in bone marrow, and at lower levels in spleen and thymus. In bone marrow, it was detected in Gr-1⁺ granuloid cells abundantly as expected and at much lower levels in the lineage marker-negative immature cells and in B220⁺ B lymphocyte lineage cells. Unexpectedly, it was also abundantly expressed in TER119⁺ erythroid cells. Both Gr-1⁺ cells and TER119⁺ cells secreted lipocalin 2 in the culture supernatant when they were cultured in vitro.

2. Specific binding of lipocalin 2 to the surface of erythroid progenitor cells
We examined the expression of a putative receptor for lipocalin 2 on TER119⁺ cells and other cell populations in the bone marrow using FACS analysis. We observed only limited binding of lipocalin 2 to TER119⁺ cells or Gr-1⁺ cells in normal bone marrow. We next examined the binding of lipocalin 2 to more immature erythroid progenitor cells and observed significant binding of lipocalin 2 to TER119^– immature erythroid progenitor cells.

3. Lipocalin 2 induces apoptosis of erythroid progenitor cells
The viability of immature erythroid progenitor cells cultured in the presence of lipocalin 2 was significantly reduced compared with control cells cultured in the absence of lipocalin 2. To determine whether the loss of viability was resulted from the induction of apoptosis in these cells, we examined caspase activity in these cells using the Caspase-Glo^TM 3/7 assay Kit. In all cases, the cells cultured in the presence of lipocalin 2 showed a higher caspase 3/7 activity than the control cells. An annexin V binding assay indicated that the loss of viability was resulted from the induction of apoptosis.

4. Lipocalin 2 inhibits differentiation of erythroid progenitor cells
CD71 is the transferrin receptor and it is well established that the vast majority of TER119^–CD71⁺ cells in the hematopoietic tissues of animals suffering acute anemia is erythroid progenitor cells. TER119^–CD71^– and TER119^–CD71⁺ cells harvested from the spleen of anemic mouse were sorted by FACS (Fig. 1 ), and cultured under conditions that allowed them to differentiate into mature erythroid cells. TER119^–CD71^– cells differentiate first to TER119^–CD71⁺ cells, subsequently to TER119⁺CD71⁺ cells, and then finally to TER119⁺CD71^– cells. We found that lipocalin 2 partly inhibited the differentiation of TER119^–CD71^– cells into TER119^–CD71⁺ cells, and strongly inhibited the differentiation of TER119^–CD71⁺ cells into TER119⁺CD71⁺ cells (Fig. 1) .

View larger version (34K): [in this window] [in a new window]   Figure 1. The in vitro effect of lipocalin 2 on the differentiation of immature erythroid cells. To obtain abundant immature erythroid progenitor cells, acute anemia was induced by the injection of phenylhydrazine into mice. Three days after phenylhydrazine injection, TER119–CD71–, and TER119–CD71+ cells in spleen were sorted by FACS, cultured in the presence or absence of lipocalin 2 for 24 h, and the cultured cells was analyzed by FACS. The middle figure in the left row shows the staining pattern of spleen cells with monoclonal antibodies against TER119 and CD71. The upper and bottom figures in the left row show the results of the analysis after cell sorting. Cells were cultured in the presence (+) or absence (–) of lipocalin 2, respectively. The percentages of cells appearing in each fraction in quadruplicate are shown at the right. Results shown are representative of 3 independent experiments using cells from different mice.

5. Expression of lipocalin 2 is reduced during acute anemia
We monitored the expression of lipocalin 2 in bone marrow and spleen after the induction of acute anemia. Despite the apparent accumulation of TER119⁺ cells in the spleen, the expression of lipocalin 2 was reduced in the spleen 2 days after phenylhydrazine injection, but had clearly recovered 4 days after injection. Then we analyzed the expression of lipocalin 2 in erythroid cells sorted by FACS 2 days after phenylhydrazine injection. We found that the expression of lipocalin 2 was reduced in erythroid cells from animals in which we had induced acute anemia. The reduction was most evident in cells from the spleen. In contrast, the expression of bcl-XL, a key inhibitor of apoptosis in erythroid cells, was increased under anemic conditions.

6. Injection of lipocalin 2 retards the recovery of RBC numbers after acute anemia
Since lipocalin 2 is expressed very abundantly in normal bone marrow and its expression is apparently inhibited after induction of acute anemia, we reasoned that injection of lipocalin 2 during acute anemic conditions might influence the production of RBC. Given that the expression of lipocalin 2 was largely reduced for a few days after induction of acute anemia before gradually recovering, we injected lipocalin 2 in the early phase of the acute anemia. We analyzed the peripheral blood five days after phenylhydrazine injection. The peripheral blood of lipocalin 2-injected mice showed a lower number of RBC and a lower hematocrit (Fig. 2 ) indicating that lipocalin 2 retarded the recovery of RBC numbers.

View larger version (23K): [in this window] [in a new window]   Figure 2. The in vivo effect of lipocalin 2 on recovery from acute anemia. Phenylhydrazine was injected intraperitoneally (60 mg/kg body weight) and simultaneously recombinant lipocalin 2 (150 µg/kg body weight) was injected into the tail vein of a 6-wk-old female mouse. Twenty-four and 48 h after phenylhydrazine injection, lipocalin 2 was again injected (150 µg/kg body weight). An equivalent quantity of bcl-XL protein (150 µg/kg body weight) was injected into control mice. The blood count was analyzed 5 days after phenylhydrazine injection. RBC, red blood cell (x104/mm3). Ht, hematocrit (%); Hb, hemoglobin (g/dl); MCV, mean corpuscular volume (fl); MCH, mean corpuscular hemoglobin (pg); MCHC, MCH concentration (%); WBC, white blood cell (x102/mm3); platelet (x104/mm3); C (open bar), control mice injected with bcl-XL; L (solid bar), mice injected with lipocalin 2. Values are mean ± SD (n=8). Broken lines are the means of results from normal mice (n=6) and are shown simply as a reference. *P < 0.05.

CONCLUSIONS AND SIGNIFICANCE

Here we showed that erythroid progenitor cells secreted lipocalin 2 and the survival and differentiation of erythroid progenitors were suppressed by lipocalin 2 (Fig. 3 ). Once acute anemia has occurred in animals, erythroid progenitors appear to escape from lipocalin 2-mediated suppression by a homeostatic feedback system (i.e., the expression of lipocalin 2 is reduced by certain factors, such as IL-3) (Fig. 3) . The reduced expression of lipocalin 2 in erythroid cells, most notably in the spleen, likely contributes to the prevention of apoptosis and inhibition of differentiation of erythroid progenitors, since erythroid cells are the dominant population in the spleen under conditions of anemia. It is likely that a very low level of lipocalin 2 creates an appropriate microenvironment for erythropoiesis in the spleen compared with bone marrow, and it may be one of the major reasons why splenomegaly is observed as a site of extramedullar erythropoiesis when urgent erythropoiesis is required. After recovery from acute anemia, lipocalin 2 may function to eliminate erythroid progenitors overproduced by an urgent hematopoietic response as has been proposed by Devireddy, et al. regarding homeostasis of leukocytes.

View larger version (53K): [in this window] [in a new window]   Figure 3. Schematic diagram of the hypothetical mechanisms of erythropoiesis regulated by lipocalin 2. Erythroid progenitor cells secrete lipocalin 2 under normal physiological conditions and the survival and differentiation of erythroid progenitors are suppressed by lipocalin 2. Once acute anemia has occurred in animals, erythroid progenitors appear to escape from lipocalin 2-mediated suppression by two mechanisms. First, the expression of lipocalin 2 in erythroid progenitors is reduced by certain factors, such as IL-3. Second, erythroid cells acquire resistance to lipocalin 2-mediated apoptosis by up-regulation of intracellular factors to prevent apoptosis. Humoral factors up-regulated by acute anemia, such as erythropoietin, should induce the expression of antiapoptotic molecules and make erythroid cells resistant to apoptosis.

Based on the activity of lipocalin 2 we have demonstrated here, it may be that anemia arising from pathological conditions, such as chronic inflammation, might be due, at least in part, to increased levels of lipocalin 2 secreted from expanded leukocytes and/or macrophages. It is possible that anemia arising from malignancies also may be partly due to the action of lipocalin 2 secreted from tumor cells. Inhibition of lipocalin 2 may constitute an effective therapy for such anemia.

Since the expression of lipocalin 2 is significant even in physiologically normal bone marrow, we propose that lipocalin 2 ordinarily induces apoptosis and/or inhibits differentiation of hematopoietic progenitors, so as to avoid the survival and differentiation of excess progenitor compartments than necessary for the maintenance of normal cell numbers. Once an urgent hematopoiesis is required, the expression of lipocalin 2 is reduced allowing otherwise apoptotic precursor cells to survive, differentiate, and rapidly supply terminally differentiated mature cells. This mechanism should permit the rapid production of mature cells, thus avoiding the need to wait for much more immature precursor cells, such as hematopoietic stem cells, to differentiate. The observation by Koury and Bondurant that a majority of erythroid progenitor cells are ordinarily dying by apoptosis in physiologically normal bone marrow might be partly due to the activity of lipocalin 2 in addition to insufficient EPO-mediated signals.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-3809fje;

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This Article Full Text (PDF) All Versions of this Article: 19/13/1881 05-3809fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Miharada, K.-i. Articles by Nakamura, Y. Search for Related Content PubMed PubMed Citation Articles by Miharada, K.-i. Articles by Nakamura, Y.