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The Journal of Immunology, 1998, 160: 345-350.
Copyright © 1998 by The American Association of Immunologists

Neutrophils but Not Eosinophils Are Involved in Growth Suppression of IL-4-Secreting Tumors1

Gabriele Noffz2,*, Zhihai Qin*, Manfred Kopf{dagger} and Thomas Blankenstein*

* Max-Delbrück-Centrum für Molekulare Medizin, Berlin, Germany; and {dagger} Basel Institute for Immunology, Basel, Switzerland


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results and Discussion
 References
 
Local expression of IL-4 by gene-modified tumor cells increases their immunogenicity by inducing an inflammatory response that is dominated by eosinophils. Eosinophils have been implicated as antitumor effector cells because the application of a granulocyte-depleting Ab inhibited rejection of IL-4 transfected tumors. This Ab did not discriminate between eosinophils and neutrophils and, therefore, this experiment could not exclude neutrophils as primary effector cells, whereas eosinophils were innocent bystander cells in IL-4 transfected tumors. We analyzed tumor growth suppression and granulocyte infiltration in IL-5-deficient (IL-5-/-) mice that had a deficiency of eosinophils, using two tumor lines (B16-F10 and MCA205) transfected to secrete IL-4. IL-4-expressing tumors were at least as efficiently rejected in IL-5-/- mice as in wild-type mice, despite an almost complete absence of tumor-infiltrating eosinophils. However, neutrophils were present in undiminished amounts and their depletion partially restored tumor growth. Furthermore, the growth of IL-5-secreting tumors was not impaired in either wild-type or IL-5-/- mice, even though it induced eosinophilia in both mouse strains. These findings demonstrate that eosinophils can be induced in IL-5-/- mice by exogenous IL-5 and argue against a compensatory effect of neutrophils in the absence of eosinophils. We conclude that 1) infiltration of IL-4 transfected tumors by eosinophils is completely IL-5 dependent, 2) eosinophils have no tumoricidal activity, and 3) neutrophils are responsible, at least in part, for tumor suppression.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results and Discussion
 References
 
The growth of IL-4 transfected tumors in vivo is suppressed in a rather reproducible fashion (1). It is caused by a biphasic rejection mechanism whereby a rapid inflammatory response inhibits tumor burden, allowing T cells, which are needed for complete tumor eradication in most cases, to develop (2, 3). Production of IL-4 by tumors is responsible for a rapid and marked infiltration of inflammatory cells, predominantly including macrophages and eosinophils (2, 3, 4, 5). Depletion of eosinophils by a granulocyte-specific Ab has been shown to inhibit tumor rejection. Therefore, it has been suggested that eosinophils are good candidates for antitumor effector cells (5). This was particularly intriguing, as no definite function was known for eosinophils (6) except in some pathologic situations (7). In contrast, eosinophil infiltration caused by tumors engineered to produce IL-5 failed to inhibit tumor growth (8). This experiment, however, was not able to determine whether eosinophils were innocent bystander cells in the transfected tumors or were insufficiently activated by IL-5 rather than by IL-4. Because of the abundant eosinophilic infiltration in IL-4-expressing tumors, neutrophils obviously escaped detection. Recently, Pericle et al. detected neutrophils in addition to eosinophils in IL-4-expressing tumors (9). The above-mentioned anti-granulocyte Ab depleted both subsets and, therefore, the relative contribution of either subpopulation to the antitumor response is not clear.

IL-5 is the major cytokine inducing proliferation and terminal differentiation of eosinophils (10). Its function cannot be replaced by other factors, because disruption of IL-5 or the specific IL-5 receptor in mice abolishes blood and tissue eosinophilia normally induced by parasite infection or allergic response (11, 12, 13). Using IL-5-deficient (IL-5-/-) mice, we show that neutrophils but not eosinophils are involved in suppression of IL-4-secreting tumors.


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

C57BL/6 mice were purchased from Bomholtgaard Breeding & Research Centre (Ry, Denmark). IL-5-deficient mice homozygous for targeted disruption of the IL-5 gene have been created as an inbred C57BL/6 line (11). The deficiency of the IL-5 gene was confirmed by PCR. All mice used in the experiments were 6 to 12 wk old and were sex matched.

Tumor cell lines

B16-F10 (B16)3 is a spontaneously derived malignant melanoma of C57BL/6 origin (14). MCA205 is a fibrosarcoma induced in C57BL/6 mice by treatment with methylcholanthrene (15). Both cell lines were cultured in RPMI 1640 supplemented with 10% FCS and nonessential amino acids. The culture medium of MCA205 contained additionally 50 µM 2-ME.

Vector construction and retroviral gene transfer

Murine interleukin (mIL)-4 cDNA was obtained from the IL-4-containing plasmid XEP-IL-4 (16) by PCR. The following primers containing both the start and stop signals and the BamHI recognition site were used: 5'-gca gga tcc tat tga tgg gtc tc (sense) and 5'-cgc gga tcc cta cga gta atc cat ttg (antisense). The PCR-amplified 0.42-kb IL-4 cDNA fragment was cloned in sense orientation downstream to elongation factor (EF)1{alpha}, a promoter in vector HyTk-EF1{alpha} that contains a hygromycin-thymidinkinase fusion gene as a selectable marker driven by the viral long terminal repeat (17, 18). The construct was termed HyTk-EF1{alpha}-IL-4. Retroviruses were generated as follows. The amphotropic packaging line PA317 (19) was transfected with the plasmid HyTk-EF1{alpha}-IL-4 using a eukaryotic transfection kit (Stratagene, Heidelberg, Germany) and selected with 0.5 mg/ml hygromycin. The virus-containing supernatant was used to infect {psi}2 cells (20) and then selected with hygromycin (0.5 mg/ml), resulting in {psi}2-IL-4 that produced a virus titer of 1 x 106 hygromycin-resistant colonies/ml. Retroviral infection of B16 and MCA205 was performed with supernatant of {psi}2-IL-4 cells. Cells resistant to hygromycin (0.5 mg/ml) were selected and cloned by limiting dilution. All cells were confirmed to be helper-virus free by hygromycin-resistance mobilization assay after prolonged culture. Mock transfectants with HyTk-EF1{alpha} were generated using the same procedure.

mIL-5 cDNA was obtained by PCR-amplifying the plasmid XEP-mIL-5 (our unpublished observations). The following primers, containing both the start and stop signals and the BamHI recognition site, were used: 5'-gca gcg gat cct cag cc (antisense) and 5'-ctt cgg atc cat gag aag g (sense). The 0.42-kb mIL-5 fragment was also cloned in sense orientation into the BamHI site of the vector HyTk-EF1{alpha}. The resulting plasmid HyTk-EF1{alpha}-mIL-5 was transfected into the cell line MCA205 using a eukaryotic transfection kit. Selection was performed as described above. All cells used were tested for mycoplasma and were found to be free of mycoplasma contamination.

Cytokine detection

IL-4 activity was determined by a proliferation assay with the IL-4-dependent cell line CT.4S (21). Cells were seeded at a density of 5 x 103/well in a 96-well flat-bottom microtiter plate (Costar, Cambridge, MA). Serial dilutions of recombinant IL-4- or 48-h-conditioned medium of transfected cells at a concentration of 1 x 106 cells/ml were added in triplicate. Activity was quantitated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay (22). Twenty microliters of MTT solution (5 mg/ml) was added to each well for 4 to 6 h. After removal of the medium, blue formazan crystals were solved in 100 µl DMSO and OD was determined on an ELISA reader (MR5000; Dynatech Laboratories Inc., Denkendorf, Germany) at 570 nm. One unit of mIL-4 was defined as the amount of IL-4 required to obtain half-maximal proliferation. Alternatively, IL-4 was determined by ELISA using purified anti-mIL-4 mAb and biotinylated anti-mIL-4 mAb (PharMingen, Hamburg, Germany) according to manufacturer recommendations.

IL-5 activity was determined by a proliferation assay using the IL-5-dependent cell line B13 (23) according to the procedure described above for IL-4 . One unit of mIL-5 was defined as the amount of IL-5 required to obtain half-maximal proliferation.

Analysis of tumor growth

Cells were harvested, washed three times in D-PBS, and injected s.c. into the flanks of wild-type or IL-5-/- mice in a volume of 0.2 ml. The number of B16 cells and variants applied was 1 x 105/mouse; the number of MCA205 cells and variants applied was 2.5 x 105. Tumor growth of the sex- and age-matched mice was measured with a caliper every 3 to 4 days. Tumor size was determined as the mean value of the largest diameter and the diameter at the right angle unless indicated differently. Mice bearing a tumor >=1.0 cm wide were scored tumor positive.

Analysis of tumor-infiltrating granulocytes

Two- and six-day-old parental and transfected tumors s.c. injected into wild-type or IL-5-/- mice were excised, and single-cell suspensions were prepared by mechanical disaggregation of minced tumor fragments. Cells were washed two times in D-PBS before analysis. For morphologic analysis, slides were prepared from a single-cell suspension using a cytocentrifuge (Cytospin 2, Shandon Instruments Inc., Astmoor, U.K.). Cytospins of 1 x 105 cells per slide were stained with Wright’s stain solution, and cell populations were identified and enumerated by light microscopy. For histologic analysis, tumor tissues were fixed in 7% neutral-buffered formalin and embedded in paraffin wax. Four- to five-micrometer thick sections of embedded tissues were cut and stained with hematoxylin and eosin. To distinguish between tumor-infiltrating granulocyte subsets with regard to their GR-1 expression, flow cytometry analysis was performed with directly coupled phycoerythrin rat anti-mouse GR1 mAb (IgG2b) (PharMingen). To determine the scatter parameter of granulocytes and their expression of the GR-1 Ag, neutrophils and eosinophils were isolated. For isolation of neutrophils (24), C57BL/6 mice were injected i.p. with 2 ml of 0.2% sodium caseinate, pH 7.2 (Sigma, Diesendorf, Germany). After 3 h, peritoneal exudate cells were harvested, washed twice, and analyzed by flow cytometry. Purity of the isolated neutrophils was >90% as determined by morphology. For isolation of eosinophils (25), C57BL/6 mice were injected i.p. with 2 ml of heat-inactivated human serum and, after 2 days, peritoneal exudate cells containing mainly eosinophils and macrophages were collected. Eosinophils were enriched further by negative selection using anti-Mac3 mAb and magnetic beads coated with anti-rat IgG (Dynabeads, Dynal, Hamburg, Germany). The enriched cell population was subsequently used for flow cytometry analysis.

Granulocyte depletion in vivo

IL-5-deficient mice were injected s.c. with 1 x 105 cells/mouse of the IL-4-producing B16 clone designated B16-IL4.140. Mice were treated with an mAb against GR-1 Ag (RB6-8C5, PharMingen) on days 1, 4, and 10. The Ab was applied i.p. at 0.25 mg/mouse on days 1 and 4 and at 0.5 mg/mouse on day 10. An identical dose of the control Ab (rat IgG, Sigma) was injected at the same time points.


   Results and Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
References
 
Growth of IL-4-expressing tumors is similarly suppressed in IL-5-/- and wild-type mice

A retrovirus was constructed containing the IL-4 gene under control of the EF1{alpha} promoter and used to express IL-4 in the melanoma B16 and the fibrosarcoma MCA205, which are both syngeneic to C57BL/6 mice. For B16, a bulk culture producing 60 U/ml IL-4 and three clones producing 5, 100, and 140 U/ml IL-4 were established. For MCA205, a bulk culture producing 30 U/ml of IL-4 and a clone producing 700 U/ml of IL-4 were established. Mock-transfected cells served as controls. In preliminary experiments we confirmed that IL-4 expression suppressed tumor growth in all cell lines and that tumor growth suppression directly correlated with the amount of IL-4 produced. Furthermore, we found that B16 cells producing 140 U/ml of IL-4 showed delayed tumor growth but that complete rejection was not induced, confirming the results found by others (26). The high IL-4-producing MCA205 clone was rejected in most cases, presumably because of its higher immunogenicity (26) or the higher amounts of produced IL-4, or both. Mock-transfected cells grew as tumors with similar kinetics in syngeneic mice when compared with parental cells. Subsequent experiments were done with B16-IL4.140 (140 U/ml IL-4) and MCA-IL4.700 (700 U/ml IL-4) cells. B16-IL4.140 and MCA-IL4.700 cells were injected in parallel with the mock-transfected cells into wild-type and IL-5-/- mice and tumor growth was compared. The IL-5-/- strain was generated by targeting the IL-5 gene in C57BL/6 cells and breeding embryonic stem cell germline chimeras with C57BL/6 mice. Heterozygous IL-5+/- mice were crossed two generations further onto C57BL/6 mice before breeding to homozygosity and, therefore, can be considered inbred. As shown in Figure 1Go, the growth kinetics of B16-IL4.140 and MCA205-IL4.700 were very similar in wild-type and IL-5-deficient mice. While mock-transfected cells rapidly gave rise to tumors in all cases, growth of B16-IL4.140 cells was strongly suppressed. With a delay of about 2 mo, cells grew out as tumors in both mouse strains. Previously, we had found that the late outgrowth of IL-4-transfected tumor cells was associated with loss of IL-4 production (3). Similarly, two of two B16-IL4.140 tumors in IL-5+/+ mice and three of three tumors in IL-5-/- mice isolated between days 60 and 100 after tumor cell injection had either completely stopped or drastically reduced IL-4 production. MCA205-IL4.700 cells were rejected in almost all cases, regardless of the mouse strain. Tumor growth suppression by IL-4 seemed slightly more efficient in IL-5-/- than in wild-type mice, the meaning of which is not yet clear. Tumor growth inhibition was also similarly suppressed by IL-4 production when bulk cultures of IL-4 gene-transduced B16 cells were used. This suppression rules out the possibility of clonal artifacts (data not shown). We conclude that IL-5 is not important for IL-4-mediated tumor growth inhibition.



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  		FIGURE 1. Growth of IL-4-secreting tumors is similarly suppressed in IL-5+/+ and IL-5-/- mice. A, Wild-type C57BL/6 (closed symbols) and IL-5-/- mice (open symbols) (n = 5) were injected s.c. with 2.5 x 105 MCA-IL4.700 (circles) or MCA205 mock (squares) cells and tumor incidence observed. One of two experiments with similar results is shown. B, IL-5+/+ (closed symbols) and IL-5-/- mice (open symbols) were injected s.c. with 1 x 105 B16-IL4.140 (circles) or B16 mock (squares) cells and tumor incidence observed. One of two experiments with similar results is shown (n = 4–6).

 
Absence of eosinophils in IL-4-expressing tumors of IL-5-/- mice reveals a strong infiltration of neutrophils involved in tumor suppression

The IL-5-/- mice are deficient in mature eosinophils but otherwise seem normal. The undiminished antitumor response in IL-5-/- mice may simply indicate that eosinophils are not involved in tumor rejection. However, IL-5-/- mice have a residual pool of basal eosinophils. Local IL-4 expression in conjunction with chemokines (monocyte chemotactic protein-5, macrophage inflammatory protein-1{alpha}, RANTES, eotaxin) may allow trafficking, localization, and accumulation of these eosinophils to tumor sites. Indeed, it has been suggested that eotaxin secreted from endothelial cells or inflammatory cells may be able to initiate and supplement tissue eosinophilia by sequestering, into the circulation, eosinophils that are migrating through noninflamed tissues (27). Further, eotaxin is induced by IL-4-secreting tumors (28), and eotaxin expression is not abolished in inflamed tissues of IL-5-/- mice (M. Kopf, unpublished observation). To analyze the possibility that eosinophils were induced by IL-4-secreting tumors in IL-5-/- mice, tumor-infiltrating cells were characterized by morphology and immunostaining. Hematoxylin/eosin-stained tumor sections excised on days 2 and 6 revealed a negligible infiltration in B16 tumors but a dramatic leukocyte infiltration in the IL-4-secreting tumor B16-IL4.140, which was also observed in several other tumor models, e.g., J558L (3, 4), TS/A (9), and Renca (2). The same results were observed in IL-5+/+ and IL-5-/- mice. In IL-4-secreting tumors of wild-type mice, granulocytes, and macrophages were abundantly present. In IL-4-secreting tumors of IL-5-/- mice, eosinophils seemed to be much less abundant despite the use of an otherwise similar infiltrate (not shown). To determine the percentage of neutrophils and eosinophils in IL-4-transfected tumors of IL-5+/+ and IL-5-/- mice, cytospins of single-cell suspensions of reisolated tumors were prepared (Fig. 2Go, A and B) and both granulocyte subsets were quantified (Fig. 2GoC). The percentage of neutrophils in tumors of both mouse strains was quite similar (31.5% in IL-5+/+ vs 32.5% in IL-5-/- mice). In contrast, 17.8% eosinophils were found in tumors of wild-type mice whereas <1% eosinophils were present in tumors of IL-5-/- mice on day 2. Macrophages were found in both wild-type and IL-5-/- mice in large amounts. These results were confirmed by flow cytometry analysis of GR-1 expression of infiltrating granulocytes (Fig. 3Go). The Ag GR-1, which is expressed on mature granulocytes but absent from mononuclear cells, has been shown to be expressed much more strongly on neutrophils than on eosinophils (29). To confirm this, peritoneal cell populations of enriched eosinophils and neutrophils were prepared and stained for GR-1 expression. Activated neutrophils showed much brighter staining by flow cytometry analysis when compared with eosinophils (Fig. 3Go, A and B). Using these cells as controls, granulocyte populations present in reisolated IL-4-expressing tumors of IL-5+/+ and IL-5-/- mice were stained for GR-1 expression. In tumors of IL-5+/+ mice (Fig. 3GoC), a large number of cells staining brightly for GR-1 were identified as neutrophils (compare with Fig. 3GoA). Additionally, a second peak of low GR-1 expressing cells was identified as eosinophils (compare with Fig. 3GoB). In tumors of IL-5-/- mice, neutrophils were present in similar amounts, but eosinophils were almost completely absent (Fig. 3GoD). Therefore, we conclude that 1) eosinophils appear in IL-4-secreting tumors in a strictly IL-5-dependent fashion, and 2) eosinophils are not needed for growth suppression of IL-4-transfected tumors.



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  		FIGURE 2. Eosinophils but not neutrophils are absent in IL-4-secreting tumors of IL-5-/- mice. Five x 106 B16-IL4.140 cells were injected into IL-5+/+ and IL-5-/- mice. Tumors were excised on day 2, disaggregated, and spun onto slides. Cytospin preparations were stained with Wright’s stain solution and enumerated. Examples of tumor infiltrating cells in IL-5-/- (A) and IL-5+/+ mice (B) are shown (N, neutrophils; E, eosinophils; M, macrophages). C, Differential counts were performed using the same cytospin preparation and percentages of eosinophils (closed bars) and neutrophils (open bars) are shown. Data represent the mean ± SD from three distinct fields of 200 cells and are representative of three independent experiments.

 


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  		FIGURE 3. Eosinophils (E) but not neutrophils (N), identified by their different expression level of GR-1, are absent in IL-4-secreting tumors of IL-5-/- mice. Peritoneal neutrophils (A) and eosinophils (B) were enriched as described in Materials and Methods, stained with anti-GR1 mAb, and 30,000 granulocytes were gated by size and granularity specific for neutrophils and eosinophils, respectively. Therefore, neutrophils can be distinguished from eosinophils by their stronger GR-1 expression. Using these cells as controls or with an isotype-matched mAb (C), tumor-infiltrating granulocytes in IL-4-secreting tumors were analyzed. Five x 106 B16-IL4.140 cells were injected s.c. into IL-5+/+ (C) and IL-5-/- mice (D), isolated 2 days later, and analyzed for granulocyte infiltration. Granulocytes (eosinophils and neutrophils) were gated by scatter parameters and GR-1-positive cells were determined. Neutrophils (high GR-1 expression) are present in both mouse strains but eosinophils (low GR-1 expression) are present only in IL-5+/+ (C) but not in IL-5-/- mice (D).

 
It has been demonstrated in several tumor models, including one used in this study (B16), that granulocyte depletion using the GR-1-specific Ab RB6-8C5 at least partially restored growth of IL-4-secreting tumors in wild-type mice (5, 9). The absence of eosinophils and presence of neutrophils in IL-4-secreting tumors of IL-5-/- mice suggested that neutrophils are responsible for tumor growth suppression. To confirm this, B16-IL4.140 cells were injected into IL-5-/- mice depleted of granulocytes by RB6-8C5 mAb. On day 18, three of five IL-5-/- mice developed a tumor nodule (Fig. 4Go) similar in size to that observed by Tepper et al. (5). Histologic examination of the tumors revealed strongly reduced numbers of infiltrating cells but large numbers of viable tumor cells comparable with parental tumors (not shown). No tumors were detected at that time point in IL-5-/- mice injected with an isotype-matched control mAb (0/5). We conclude that neutrophils belong to the primary cells leading to rejection of IL-4-transduced tumors. At the present time, it is not clear whether neutrophils contribute to tumor suppression by virtue of their regulatory or tumoricidal activity. Granulocyte depletion could not completely restore tumor growth as compared with in vivo anti-IL-4 mAb experiments (3, 4, 5), indicating that other effector cells, such as macrophages (30), also contribute to IL-4-induced tumor suppression. Even though neutrophils have been shown to have direct tumoricidal activity against IL-2-secreting tumors (31), it is not clear in our system whether their involvement in tumor suppression results from a direct or an indirect effect. In this regard, it is interesting to note that granulocyte depletion by RB6-8C5 mAb restored growth of tumors that were transfected to secrete a variety of different cytokines, e.g., IL-2 and TNF (32). This could mean that the different cytokines induce neutrophils in a redundant fashion or, alternatively, that secondary-host cytokines, e.g., granulocyte-CSF (33), are induced in other inflammatory cells by the cytokine gene-modified tumor cells which then activate neutrophils for antitumor activity.



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  		FIGURE 4. Granulocyte depletion partially restores growth of B16-IL4.140 cells in IL-5-/- mice. One x 105 B16-IL4.140 cells were injected s.c. into IL-5-/- mice that were depleted in parallel for granulocytes by i.p. injection of mAb RB6-8C5 (squares) in a total amount of 1 mg over a period of 10 days. The control group was identically treated with an isotype-matched control mAb (circles). *, Number of mice with tumor/number of mice in experiment on day 18.

 
IL-5-expressing tumors induce eosinophilia in IL-5-/- mice but grow progressively

Because the failure to detect eosinophils in IL-4-secreting tumors of IL-5-/- mice could be due to the fact that the residual pool of eosinophils could not be induced, we determined whether eosinophil infiltration was observed if one of the investigated tumors produced IL-5 instead of IL-4. This being the case, it also would allow confirmation that eosinophils attracted to IL-5-secreting tumors would not suppress tumor growth (8). MCA205 cells were transfected with the IL-5 gene, resulting in MCA205-IL5. IL-5-/- and wild-type mice were injected with 2.5 x 105 cells/mouse of the bulk culture producing about 100 U/ml IL-5. Analysis of the tumor growth showed no change in the tumorigenicity between parental and IL-5-transduced tumor cells in either wild-type or IL-5-/- mice (Fig. 5Go). Granulocyte infiltration of IL-5-secreting tumors was analyzed on day 20. Eosinophilic infiltration was observed not only in IL-5+/+ but also in IL-5-/- mice (Fig. 6Go). Neutrophils were present in the IL-5-secreting tumors of both mouse strains only in small amounts. Because of the progressive growth of the tumors and the accumulating release of IL-5, blood eosinophilia was detected in both strains (19.3% in IL-5-/- mice vs 15.6% in IL-5+/+ mice). We conclude that 1) the generation and recruitment of eosinophils can be induced in IL-5-/- mice by IL-5 application and 2) IL-5 induced eosinophils do not have tumoricidal activity. We cannot completely exclude the possibility that neutrophils compensate for eosinophils in IL-4-mediated tumor suppression in IL-5-/- mice. This seems unlikely, however, because 1) the accumulation of eosinophils in IL-4-secreting tumors is mediated by IL-5 that should derive from an unidentified cellular source induced by IL-4, 2) neutrophils occur in similar amounts in IL-4-secreting tumors of IL-5+/+ and IL-5-/- mice, and 3) no direct biologic effect of IL-4 on eosinophils has been demonstrated.



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  		FIGURE 5. IL-5 secretion by tumor cells does not inhibit tumor growth. IL-5+/+ (solid symbols) and IL-5-/- mice (open symbols) were injected s.c. with 2.5 x 105 MCA205-IL5 (circles) or MCA205 cells (squares). Percentage of tumor-free mice (n = 5) is shown.

 


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  		FIGURE 6. Eosinophils are present in IL-5-secreting tumors from both IL-5+/+ and IL-5-/- mice. Mice were injected s.c. with 2.5 x 105 MCA205-IL5 cells and tumors were excised on day 20. Differential counts were performed by cytospin preparation of tumor infiltrating cells, and percentage of eosinophils (closed bars) and neutrophils (open bars) are shown. Data represent the mean ± SD from five distinct fields of 100 cells and are representative of two independent experiments.

 
The evolutionary role of eosinophil remains far from clear. Generally, eosinophilic responses are associated with helminth infection. However, eosinophil depletion by IL-5 neutralization or IL-5 gene disruption did not ablate protective immunity against Schistosoma mansoni, Heligmosomoides polygyrus, Nippostrongylus brasiliensis, and Mesocestoides corti (11, 34), whereas the survival of Angiostrongylus cantonensis is enhanced in brains of IL-5-receptor {alpha}-chain (IL-5R{alpha})-deficient mice (13). Our results put into question the role of eosinophils in an antitumor response. The function of this evolutionary conserved cell type needs to be analyzed further.


   Acknowledgments
 
We thank M. Rösch for excellent technical assistance and S. Lupton for providing the vector tgLS(+)HyTk.


   Footnotes
 
1 This work was supported by grants from The Deutsche Krebshilfe, Mildred Scheel Foundation, and Bundesministerium für Bildung und Forschung, Bonn, Germany (BMBF 01 KV 9506/2 and BMBF 01 EC 9406,Q1). The Basel Institute for Immunology was founded and is supported by Hoffmann-La Roche. Back

2 Address correspondence and reprint requests to Gabriele Noffz, Max-Delbrück-Centrum für Molekulare Medizin Robert-Rössle-Strasse 10, 13122 Berlin, Germany. E-mail address: Back

3 Abbreviations used in this paper: B16, B16-F10 (tumor cell line); mIL, murine interleukin; EF, elongation factor; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. Back

Received for publication July 8, 1997. Accepted for publication September 19, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 References
 

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