The expression profiles of chemokines, innate immune and apoptotic genes in tumors caused by Rous Sarcoma Virus (RSV-A) in chickens.

Innate immune genes play an important role in the immune responses to Rous sarcoma virus (RSV)-induced tumor formation and metastasis. Here, we determined in vivo expression of chemokines, innate immune and apoptotic genes in Synthetic Broiler Dam Line (SDL) chickens following RSV-A infection. The mRNA expression of genes was determined at the primary site of infection and in different organs of progressor, regressor and non-responder chicks, using RT-qPCR. Our results indicated a significant upregulation of: (1) chemokines, such as MIP1ß and RANTES, (2) the innate immune gene TLR4, and (3) p53, a tumor-suppressor gene, at the site of primary infection in progressor chickens. In contrast, inducible nitric oxide synthase (iNOS) gene expression was significantly downregulated in progressor chicks compared to uninfected, control chicks. All of the innate immune genes were significantly upregulated in the lungs and liver of the progressor and regressor chicks compared to control chicks. In the spleen of progressor chicks, RANTES, iNOS and p53 gene expression were significantly increased, whereas MIP1ß and TLR4 gene expression was significantly downregulated, compared to control chicks. The lungs and livers of non-responder chicks expressed a low level of iNOS and MIP1ß, whereas RANTES, TLR4, and p53 gene expression were significantly upregulated compared to uninfected control chicks. In addition, there was a significant downregulation of RANTES, MIP1ß, and TLR4 gene expression in non-responder chicks. These results suggest the different response to infection of chicks with RSV-A is due to differential changes in the expression of innate immune genes in different organs.

MHC-B haplotypes impact susceptibility and resistance to RSV-A infection.

We investigated the impact of haplotype of major histocompatibility complex (MHC)-B on the outcome of infection of Synthetic Dam Line (SDL) broiler strain with Rous Sarcoma Virus (RSV). Genomic analysis of MHC-B haplotypes, revealed a total of 12 known standard haplotypes that constituted to twenty-five different genotypes and one new haplotype of 217 bp size, designated BX. The inoculation of RSV-A in SDL chicks resulted in the development of tumors of progressive or regressive phenotypes with varying tumor profile index (TPI). Haplotypes B2, B21 and B22had low TPI scores (1 or 2) with less mortality and were resistant to RSV-A tumor. The haplotypes B13, B13.1., B15, B15.1. and B15.2. had significantly higher TPI scores (5 or 6), indicating a susceptibility to RSV-A. The genotype, Bx /Bx, had a mean TPI score of 3.67 ± 1.33, which was closer to the resistant haplotype. Sequence analysis of the new haplotype (BX) revealed 99.5% similarity with B2 haplotype. Metastases was observed in 44% of chicks and comprised of mixed fibrosarcoma and myxosarcoma.

Apoptin, a tumor-selective killer.

Apoptin, a small protein from chicken anemia virus, has attracted great attention, because it specifically kills tumor cells while leaving normal cells unharmed. The subcellular localization of apoptin appears to be crucial for this tumor-selective activity. In normal cells, apoptin resides in the cytoplasm, whereas in cancerous cells it translocates into the nucleus. The nuclear translocation of apoptin is largely controlled by its phosphorylation. In tumor cells, apoptin causes the nuclear accumulation of survival kinases including Akt and is phosphorylated by CDK2. Thereby, apoptin redirects survival signals into cell death responses. Apoptin also binds as a multimeric complex to DNA and interacts with several nuclear targets, such as the anaphase-promoting complex, resulting in a G2/M phase arrest. The proapoptotic signal of apoptin is then transduced from the nucleus to cytoplasm by Nur77, which triggers a p53-independent mitochondrial death pathway. In this review, we summarize recent discoveries of apoptin's mechanism of action that might provide intriguing insights for the development of novel tumor-selective anticancer drugs.

Anti-neoplastic effect of chicken anemia virus VP3 protein (apoptin) in Rous sarcoma virus-induced tumours in chicken.

The anti-neoplastic effect of chicken anemia virus VP3 protein (apoptin) was investigated in vitro in Rous sarcoma virus (RSV)-transformed chicken embryo fibroblast (CEF) cells and in RSV-induced tumours of specific-pathogen-free (SPF) chicks in vivo. The apoptin gene was cloned in the pVAX expression vector and in vitro expression of the recombinant vector pVAX-CAV-VP3 was confirmed. Two groups of SPF chicks, each containing ten chicks, were used. Chicks in groups I and II were inoculated with RSV at 1 day old. Group I served as the control, receiving pVAX vector without insert, and group II received recombinant vector pVAX-CAV-VP3 containing the apoptin gene, on day 10. An in vitro study confirmed that apoptin induced apoptosis in RSV-transformed CEF cells, which was demonstrated by observation of the characteristic changes of apoptosis using the indirect immunofluorescence technique and acridine orange/ethidium bromide staining. In vivo study also indicated that apoptin induced apoptosis and caused tumour regression by an intratumoral-delivery method. Apoptotic changes, such as nuclear condensation, fragmentation of the chromatin and formation of apoptic bodies in the tumour cells, were demonstrated by histopathology and acridine orange/ethidium bromide staining. No apoptotic changes were seen in the tumours of the control group. The results of the present study showed that apoptin had an anti-neoplastic effect in vivo and in vitro in RSV-induced tumours. The anti-neoplastic effect is due to apoptin-induced apoptosis. Further improvements in the dose, delivery method and delivery frequency of the apoptin-expressing recombinant vector could help to develop apoptin as an anti-neoplastic drug.

Arising podosomal structures are associated with neoplastic cell morphological phenotype induced by the microenvironment.

Increased numbers of rosettes of podosomes were observed in overgrown rat Rous sarcoma RsK4 cells. A possible role of these structures in nutrient uptake in tumour cell survival was investigated by exposure to acute starvation. A single cell suspension of RsK4 cells in Hanks balanced salt solution was allowed to interact with either clean uncoated or serum-coated for bait coverglasses. Confocal microscopy revealed contrasting 3D cell morphologies that were associated with conspicuous patterns of podosomal structures, which on the coated coverglasses resembled the sealing zones of osteoclasts, while on the uncoated coverglasses they resembled the marginal podosomes of migrating monocyte-derived cells. Thus, the arising podosomal structures, the involvement of which in an uptake of nutrients appeared feasible morphologically, were associated with the emerging 3D cell shapes guided by the microenvironment. Such phenotypic plasticity of neoplastic RsK4 cells in response to microenvironmental challenge suggested that uniqueness in cellular attributes within the neoplastic cell population could be crucial for the malignant potential.

Genetic analysis of the growth curve of Rous sarcoma virus-induced tumors in chickens.

White Leghorn chicks homozygous for B19 MHC haplotype were selected for 18 generations on tumor regression after inoculation in the wing web with an SR-D strain of Rous sarcoma virus (RSV) at 4 wk of age. Each chick was assigned a tumor profile index (TPI) based on age at death and size of the tumor. During 18 generations, 2,010 birds were divergently selected on TPI for either progression or regression of the tumor (P and R lines). A Brody growth curve was fitted for each bird. Brody function parameters included the asymptotic tumor volume (A), the factor for increased growth in progression phase (K1), the factor for decreased growth in regression phase (K2), age at maximum volume (Tmax), and maximum volume of the tumor (Vmax). Tumor growth curves were found to be different according to line, sex, and restriction fragment pattern Y complex Rfp-Y MHC haplotype (Yw*15, Yw*16, and Yw*17). Within the P line, birds from the Yw*16 haplotype reached Vmax at an earlier age than Yw*15 and Yw*17, but with a lower Vmax value. Within the R line, tumor growth curves of birds from Yw*16 and Yw*17 haplotypes were similar. Rank correlations between the different parameters and TPI were low (between -0.26 and 0.36). Heritability estimated by the sire component was high for Vmax (0.73). Heritabilities of Tmax and K2 were moderate (0.20 to 0.23 for Tmax and 0.18 to 0.21 for K2) allowing these traits to be used as selection criteria. Heritabilities of A and K1 were lower than 0.12. Modeling the growth curve should contribute to better distinction between progressors and regressors.

Major histocompatibility (B) complex control of responses against Rous sarcomas.

The chicken major histocompatibility (B) complex (MHC) affects disease outcome significantly. One of the best characterized systems of MHC control is the response to the oncogenic retrovirus, Rous sarcoma virus (RSV). Genetic selection altered the tumor growth pattern, either regressively or progressively, with the data suggesting control by one or a few loci. Particular MHC genotypes determine RSV tumor regression or progression indicating the crucial B complex role in Rous sarcoma outcome. Analysis of inbred lines, their crosses, congenic lines, and noninbred populations has revealed the anti-RSV response of many B complex haplotypes. Tumor growth disparity among lines identical at the MHC but differing in their background genes suggested a non-MHC gene contribution to tumor fate. Genetic complementation in tumor growth has also been demonstrated for MHC and non-MHC genes. RSV tumor expansion reflects both tumor cell proliferation and viral replication generating new tumor cells. In addition, the B complex controls tumor growth induced by a subviral DNA construct encoding only the RSV v-src oncogene. Immunity to subsequent tumors and metastasis also exhibit MHC control. Genotypes that regressed either RSV or v-src DNA primary tumors had enhanced protection against subsequent homologous challenge. Regressor B genotypes had lower tumor metastasis compared with progressor types. Together, the data indicate that B complex control of RSV tumor fate is strongly defined by the response to a v-src-determined function. Differential RSV tumor outcomes among various B genotypes may include immune recognition of a tumor-specific antigen or immune system influences on viral replication.

Multidrug-resistant tumor cells with decreased malignancy: a role for integrin alphavbeta3.

We studied whether acquisition of multidrug resistance (MDR) by tumor cells can alter their integrin profile and malignant behavior. Hamster fibroblast cell line HET-SR-2SC-LNM was selected for MDR, yielding the 2SC/20 subline. Compared with the parental cells, the 2SC/20 subline weakly adhered to denatured collagen (dCol) which correlated with decreased expression of alphavbeta3, a dCol receptor. Importantly, 2SC/20 subline demonstrated significantly decreased activity of collagenase MMP-2, lower ability to invade Matrigel, and attenuated metastasis in syngeneic animals. We provide evidence for the first time that selection for MDR can be associated with down-regulation of alphavbeta3 integrin, supporting our recent proof of the pro-apoptotic role of this integrin (Oncogene 20 (2001) 4710). Lack of alphavbeta3 expression may link cell survival under toxic conditions with decreased malignancy of the resulting drug resistant tumor.

Nonmajor histocompatibility complex alloantigen effects on the fate of Rous sarcomas.

Rous sarcoma virus-induced tumor outcome is controlled by the MHC (B). Additional data, using controlled segregation in families, has indicated non-MHC effects as well, but few studies have focused on blood groups other than the B complex. Segregating combinations of genes encoding erythrocyte (Ea) alloantigen systems A, C, D, E, H, I, P, and L in B2B5 and B5B5 MHC (B) backgrounds were examined for their effects on Rous sarcomas. Six-week-old chickens were inoculated in the wing-web with 30 pfu of Rous sarcoma virus (RSV). Tumors were scored six times over a 10-wk period. A tumor profile index (TPI) was assigned to each chicken based on the six tumor size scores. Response was evaluated using tumor size at each measurement period, TPI, and mortality. The genotypes of Ea systems A, C, D, E, H, I, and P had no significant effect on any parameter in either B complex population. The Ea-L system had an effect on Rous sarcomas in the B2B5 intermediate responders and B5B5 progressors. Tumor size, TPI, and mortality were all significantly lower in B2B5 L1L1 chickens than in B2B5 L1L2 chickens. Mortality was lower in the B5B5 L1L1 birds than in B5B5 L1L2 chickens. It appears that the Ea-L system, or one closely linked, is acting in a manner independent of the B complex in response to RSV challenge.

Allelic complementation between MHC haplotypes B(Q) and B17 increases regression of Rous sarcomas.

Major histocompatibility (B) complex haplotypes B(Q) and B17 were examined for their effect on Rous sarcoma outcome. Pedigree matings of B(Q)B17 chickens from the second backcross generation (BC2) of Line UCD 001 (B(Q)B(Q)) mated to Line UCD 003 (B17B17) produced progeny with genotypes B(Q)B(Q), B(Q)B17, and B17B17. Six-week-old chickens were injected with subgroup A Rous sarcoma virus (RSV). The tumors were scored for size at 2, 3, 4, 6, 8, and 10 weeks postinoculation. A tumor profile index (TPI) was assigned to each bird based on the six tumor scores. Two experiments with two trials each were conducted. In Experiment 1, chickens (n = 84) were inoculated with 30 pock-forming units (pfu) RSV. There was no significant B genotype effect on tumor growth over time or TPI among the 70 chickens that developed tumors. Chickens (n = 141) were injected with 15 PFU RSV in Experiment 2. The B genotype significantly affected tumor growth pattern over time in the 79 chickens with sarcomas. The B(Q)B17 chickens had the lowest TPI, which was significantly different from B17B17 but not B(Q)B(Q). The data indicate complementation because more tumor regression occurs in the B(Q)B17 heterozygote than in either B(Q)B(Q) or B17B17 genotypes at a 15 pfu RSV dose and significantly so compared to B17B17. By contrast, the 30 pfu RSV dose utilized in the first experiment overwhelmed all genotypic combinations of the B(Q) and B17 haplotypes, suggesting that certain MHC genotypes affect the immune response under modest levels of viral challenge.

Protective effects of type I and type II interferons toward Rous sarcoma virus-induced tumors in chickens.

Growth of tumors induced by Rous sarcoma virus (RSV) is controlled by alleles at the major histocompatibility complex locus in chickens, indicating that immunological host defense mechanisms play a major role. We show here that the resistance phenotype of CB regressor chickens can be partially reverted by treating the animals with a monoclonal antibody that neutralizes the major serotype of chicken type I interferon, ChIFN-alpha. Injection of recombinant ChIFN-alpha into susceptible CC progressor chickens resulted in a dose-dependent inhibition of RSV-induced tumor development. This treatment was not effective, however, in CC chickens challenged with a DNA construct expressing the v-src oncogene, suggesting that the beneficial effect of type I interferon in this system resulted from its intrinsic antiviral activity and probably not from indirect immunmodulatory effects. By contrast, recombinant chicken interferon-gamma strongly inhibited tumor growth when given to CC chickens that were challenged with the v-src oncogene, indicating that the two cytokines target different steps of tumor development.

A low incidence of histiocytic sarcomatosis associated with infection of chickens with the HPRS-103 strain of subgroup J avian leukosis virus.

Ten cases of histiocytic proliferative lesions in meat-type chickens associated in low incidence with infection by subgroup J avian leukosis virus (ALV) are described. Six were field cases in adult chickens from naturally infected flocks and four were from younger birds from transmission experiments with HPRS-103 ALV or the related acutely transforming ALV strains 17 and 879. The lesions were observed mostly in the spleen and in some cases in other organs. Microscopically, the lesions were comprised mainly of pleomorphic histiocyte-like cells admixed with variable numbers of lymphoid cells. More detailed studies were carried out on two birds at 4 and 7 wk of age following infection with HPRS-103 at 1 day of age. These birds had multiple small nodular lesions in the spleen, liver, and kidney that appeared similar cytologically to the more extensive lesions in older birds. Monoclonal antibodies specific for various lymphoid and nonlymphoid accessory cells were used in immunohistochemical studies to identify a predominance of cells of monocyte/macrophage lineage, and CD4- and CD8-positive lymphocytes, in the splenic nodules. Ultrastructural studies also revealed a similar mixed population of cells. Expression of ALV group-specific antigen, and gag and ALV-J env RNA, was not a marked feature of the histiocytic lesions. The proliferative histiocytic lesion is designated a histiocytic sarcomatosis.

Influence of the transduced 3'UTR of the c-src oncogene on tumour growth induced by the v-src gene of avian sarcoma virus PR2257.

Avian sarcoma virus PR2257 contains 952 bp transduced from the left part of the 3'UTR of the chicken c-src oncogene. Deletion mutants were constructed to determine the effect of the 3'UTR on tumorigenicity in vivo and in vitro. In the presence of the 3'UTR, tumours were 3.4 times larger in vivo, and tumorigenicity was increased 2.5-fold in vitro. Several regulatory submotifs were also found within the 3'UTR. Parts of the 3'UTR were cloned into the LTR CAT plasmid and analysed for CAT expression. A 170 bp element was found to be responsible for the enhanced expression of the CAT gene. These results demonstrate the effect of the transduced 3'UTR sequence during long-term interaction between PR2257 virus and the chicken genome, and suggest a novel regulatory mechanism of the src oncogene.

Two tumor-associated membrane antigens defined by monoclonal antibodies in a transplantable sarcoma induced by Rous sarcoma virus in rat.

Two hybridoma clones have been produced by hybridization of murine myeloma cell line PAI and splenocytes from BALB/c mice immunized with cells from a transplantable sarcoma induced in rat by SR-RSV. The antibody produced by hybridoma clone 2C2 was of subclass IgG3 and recognized a cell surface antigen of 52 kD. It only cross-reacted with cells from SR-RSV-induced sarcoma in hamster, but not with cells from the chicken RSV-induced sarcoma, nor with a number of methylcholanthrene sarcomas and various other tumors of viral or other etiology developed in rats, mouse, hamsters or chickens. The antibody produced by hybridoma clone 5G2 was of subclass IgG2A and recognized an antigen of 28 kD which was located under the plasma membrane, particularly in the cell protrusions and microvilli. Cross-reactions were found with all sarcoma cells tested, indicating that this antigen might represent a common sarcoma antigen of comparatively low molecular mass.

B-complex recombinants and sarcoma regression: role of B-L/B-F region genes.

The anti-sarcoma response of three B complex recombinant haplotypes BR1(F24-G23), BR2(F2-G23), and BR3(F2-G23) was investigated. In a preliminary experiment, one male heterozygous for the BR1 recombinant haplotype and another heterozygous for the BR2 recombinant haplotype were each mated to females, some of which carried the respective recombinant. The anti-sarcoma response of progeny carrying the BR1 recombinant differed significantly from that of progeny carrying the BR2 recombinant. Subsequently, each of the three recombinant haplotypes was placed on each of four B haplotype complex backgrounds, and compared to B-G and B-L/B-F region controls on the same background haplotype. For each recombinant, significant differences in tumor growth were found between the recombinant and B-L/B-F control chickens on either one, two, or three of the four genetic backgrounds tested. For each recombinant, no differences were found between chickens carrying the recombinant and B-G region controls, which is further evidence that the gene(s) controlling Rous sarcoma growth lies in or near the B-L/B-F chromosomal region. Moreover, although the BR2 and BR3 recombinants appear to be identical serologically, they differed significantly in tumor growth suggesting that the two haplotypes are genetically different.

Inflammation is responsible for the development of wound-induced tumors in chickens infected with Rous sarcoma virus.

When newly hatched chicks are given injections of Rous sarcoma virus, a tumor develops at the site of injection. In spite of the presence of the virus in the blood, no other tumors are found distant from the site of inoculation during the life span of the animal (4-6 weeks). However, if a wound is made away from the primary tumor, a tumor develops at the site of wounding. Work in our laboratory showed previously that these wound tumors do not develop as a result of metastasis, therefore, factors released upon wounding must contribute to the development of the wound tumors. In particular, we showed that transforming growth factor (TGF) beta, a growth factor implicated in wound healing, can replace wounding in tumor development. However, we also showed that epidermal growth factor and TGF-alpha, growth factors that also have roles in wound healing, do not induce tumors. To identify the critical event(s) and to determine the mechanism involved in wound tumor development, we have continued these studies. Here we show that: (a) wound tumor development correlates with the presence of circulating virus and inflammation; (b) the virus is present in serum and in heterophils of the peripheral blood; (c) cell division at the site of wounding precedes the expression of viral proteins; (d) in addition to TGF-beta, acidic and basic fibroblast growth factors can also replace wounding in tumor development; (e) these three factors (TGF-beta, acidic fibroblast growth factor, basic fibroblast growth factor) which promote tumors also induce inflammation, whereas epidermal growth factor and TGF-alpha do not; and (f) during the inflammatory response, blood vessel leakage occurs as tested by the release of fibrinogen into the tissues. To test the possibility that inflammation is the key element in the development of these wound tumors, we used beta-methylprednisolone, an antiinflammatory drug that inhibits inflammation (including blood vessel leakage), to determine if wound tumor development could be prevented. We found that when inflammation was inhibited, tumors were also inhibited; when inflammation could not be stopped, tumors developed as before. These results indicate that the effect of wounding on the development of wound tumors in Rous sarcoma virus-infected chicks is accomplished through the cytokines released by the inflammatory cells at the site of wounding. These inflammatory mediators play a critical role in providing the conducive environment for oncogene integration and activation, and subsequent development of tumors.(ABSTRACT TRUNCATED AT 400 WORDS)

Response of six major histocompatibility (B) complex recombinant haplotypes to Rous sarcomas.

Six B complex recombinants, BR1 (F24-G23), BR2 (F2-G23), BR3 (F2-G23), BR4 (F2-G23), BR5 (F21-G19), and BR6 (F21-G23), from the fourth backcross generation to highly inbred line UCD 003 (B17B17) were studied for their response to Rous sarcomas. Eight hatches were produced from heterozygous (BRnB17) parents. Chicks were wingweb inoculated with 50 pock-forming units of Rous sarcoma virus (RSV) at 6 wk of age. A tumor profile index (TPI), based on degree of tumor regression, was evaluated by analysis of variance. BR2, BR3, and BR4 are serologically similar F2-G23 recombinants. Haplotype B2, the origin of BF2, is a known tumor regressor, yet BR2BR2 chickens had a significantly lower TPI than BR3BR3 and BR4BR4 chickens. The TPI of BR2BR2 (F2-G23) chickens was also significantly lower than the TPI of chickens homozygous for BR1 (F24-G23) and BR5 (F21-G19). The BR6BR6 (F21-G23) chickens had significantly lower TPI than all homozygotes except BR2BR2 (F2-G23). Among heterozygous genotypes, BR2B17, BR5B17, and BR6B17 differed significantly from BR1B17, BR3B17, and BR4B17. These results suggest that serologically similar recombinants that contain (F2-G23) possess different genes affecting tumor regression. In addition, degrees of tumor regression in BR5 (F21-G19) and BR6 (F21-G23), both of which contain BF21, may be due to genetic differences within the B-F/B-L or B-G regions.

Immunohistochemical analysis of cells infiltrating Rous sarcoma virus-induced tumors in chickens.

The aim of our experiments was the analysis of peripheral blood lymphocytes (PBL) and tumor infiltrating lymphocytes (TIL) in chicken inbred congenic lines, CB line with regressing and CC line with progressing RSV-induced sarcomas. For serological analysis, monoclonal antibodies to CD4, CD8, TCR1, TCR2, MHC class I and class II antigens were used. Significant differences determined by flow cytometry in CD4+ and CD8+ populations of PBL between CB chickens with tumors in progressive phase and CC chickens were not confirmed by means of TIL analysis. TIL were analysed either in suspension prepared from tumors by means of flow cytometry or on cryostat section of tumors. But using the histochemical method we observed more non-specific esterase positive cells in cryostat sections from CB than CC tumors. The role of macrophages in the regression of RSV-induced tumors in CB chickens has to be further analysed.

Immune-based resistance to the formation of v-src-induced distal tumors.

Although v-src, the oncogene of Rous sarcoma virus, has been shown to be capable of inducing lethal tumors at visceral sites distal to the primary tumor mass, the mechanisms opposing visceral tumor formation remain to be elucidated. In the present study, we show that visceral tumors, many of which represent a metastasis spawned by the primary mass, are found only in hosts exhibiting reduced levels of tumor immunity. We conclude that it is the weakness of the tumor immune response, rather than a lack of expression of tumor antigen on visceral tumor cells, that is a major underpinning of the formation of v-src-induced visceral tumors.

Analysis of macrophage functions in Rous sarcoma-induced tumor regressor and progressor 6.B congenic chickens.

Macrophage functional competence was studied in two congenic chicken lines 6.6-2 (B2B2) and 6.15-5 (B5B5) which are regressors and progressors, respectively, of Rous sarcoma-induced tumors. Sephadex-elicited abdominal exudate cells (AEC) were harvested from 4-week-old chickens to determine their total number, glass adherence potential, percentage of adherent macrophages and phagocytosis of antibody coated (ops) and uncoated (unops) sheep red blood cells (SRBC). Tumoricidal abilities of culture medium conditioned with lipopolysaccharide treated macrophages and of macrophages cocultured with target cells were assessed against 51Cr-labelled tumor cell targets. The congenic lines did not differ in total AEC or percent macrophages. However, AEC from B5B5 birds exhibited significantly lower (P < 0.05) glass-adherence potential than AEC from B2B2 birds exhibited significantly lower (P < 0.05) glass-adherence potential than AEC from B2B2 birds. The percentage of phagocytic macrophages did not differ between lines for unop-SRBC, whereas a higher percentage of B5B5 compared with B2B2 birds (P < 0.05) macrophages phagocytized ops-SRBC. Macrophages from B5B5 birds had significantly (P < 0.05) lower activity in both tumoricidal tests. These results imply that the tumor progression in B5B5 birds is associated with reduced activation of macrophages towards a tumoricidal pathway.