• View in gallery
    Figure 1—

    Representative photomicrographs of sections of archived neoplastic (A) and nonneoplastic (B) lymph node specimens from 2 dogs illustrating the relative expression of LH receptors, as determined immunohistochemically. Negative controls for each tissue specimen (upper right inset of each panel) were treated in a similar manner except that the primary antibody was not applied. Immunohistochemical LH receptor-specific stain with hematoxylin counterstain; bar = 10 μm for main and inset images.

  • View in gallery
    Figure 2—

    Plots of the percentage of LH receptor (LHR)-positive circulating nonneoplastic T and B lymphocytes obtained from 12 healthy adult dogs, as determined by flow cytometry. Each circle represents the percentage of LHR-positive cells for 1 dog (6 dogs/subgroup). For each plot, the horizontal line in the middle represents the mean and the shorter horizonal lines at the top and bottom represent the upper and lower 95% confidence intervals for the mean. A—Data regarding T lymphocytes grouped on the basis of sex of the dogs. The percentage of LHR-positive T lymphocytes did not differ significantly (P = 0.18) between males and females. B—Data regarding B lymphocytes grouped on the basis of sex. The percentage of LHR-positive B lymphocytes did not differ significantly (P = 0.06) between males and females. C—Data regarding T lymphocytes grouped on the basis of reproductive status. The percentage of LHR-positive T lymphocytes in sexually intact dogs was significantly (P = 0.049) less than that in gonadectomized dogs. D—Data regarding B lymphocytes grouped on the basis of reproductive status. The percentage of LHR-positive B lymphocytes did not differ significantly (P = 0.45) between sexually intact and gonadectomized dogs.

  • View in gallery
    Figure 3—

    Representative scatterplot (A) and histogram (B) for the flow cytometric detection of LHR expression by T-cell lymphoma cells from an immortalized cell line derived from a dog with primary lymphoma. A—Forward (FSC) versus side scatter (SSC) plot indicating cell size and granularity, respectively, of T-cell lymphoma cells that did (green) or did not (blue) express LHR (P1 represents the percentage of lymphocytes within the gate). B—Histogram indicating the intensity of fluorescence of T-cell lymphoma cells that did (green peak) or did not (blue peak) express LHRs (P2 represents the percentage of cells that expressed LHRs, and the horizontal bar represents the range of fluorescent intensities of those cells).

  • 1. Kaiser HE. Animal neoplasms: a systematic review. In: Kaiser HE, ed. Neoplasms: comparative pathology of growth in animals, plants and man. Baltimore: Williams & Wilkins, 1981;747812.

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  • 2. Moulton JE, Harvey JW. Tumors of lymphoid and hematopoietic tissue. In: Moulton JE, ed. Tumors of domestic animals. 3rd ed. Berkeley, Calif: University of California Press, 1990; 231307.

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  • 3. Burke A. Lymphoma in dogs: symptoms, diagnosis, and treatment. American Kennel Club. Available at: www.akc.org/expert-advice/health/common-conditions/lymphoma-in-dogs-symptoms-diagnosis-and-treatment/. Accessed Apr 22, 2018.

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  • 4. Fulkerson CM. Multicentric lymphoma in the dog: an overview of diagnostics, treatment and prognosis. Available at: invma.org/wp-content/uploads/sites/5/2016/09/Lympho ma-in-the-Dog.pdf. Accessed Apr 21, 2018.

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  • 5. Umeki S, Ema Y, Suzuki R, et al. Establishment of five canine lymphoma cell lines and tumor formation in a xenotransplantation model. J Vet Med Sci 2013;75:467474.

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  • 6. Valli VE, Kass PH, Myint MS, et al. Canine lymphomas: association of classification type, disease stage, tumor subtype, mitotic rate, and treatment with survival. Vet Pathol 2013;50:738748.

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  • 7. Fan TM. Overview of canine lymphoma. Merck Veterinary Manual. 2018. Available at: www.merckvetmanual.com/circulatory-system/canine-lymphoma/overview-of-canine-lymphoma. Accessed Apr 21, 2018.

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  • 8. Teske E. Canine malignant lymphoma: a review and comparison with human non-Hodgkin's lymphoma. Vet Q 1994;16:209219.

  • 9. Dobson JM. Breed-predispositions to cancer in pedigree dogs. ISRN Vet Sci 2013;2013:941275.

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  • 11. Zink MC, Farhoody P, Elser SE, et al. Evaluation of the risk and age of onset of cancer and behavioral disorders in gonadectomized Vizslas. J Am Vet Med Assoc 2014;244:309319.

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  • 12. Beijerink NJ, Buijtels JJ, Okkens AC, et al. Basal and GnRH-induced secretion of FSH and LH in anestrous versus ovariectomized bitches. Theriogenology 2007;67:10391045.

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  • 13. Lin J, Loujin S, Lei ZM, et al. Lymphocytes from pregnant women express human chorionic gonadotropin/luteinizing hormone receptor gene. Mol Cell Endocrinol 1995;111:R13R17.

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  • 14. Rouabhia M, Deschauz P, Othmane O. Neuroimmunomodulation of the immune response by the endocrine system: effect of luteinizing hormone (LH) on the proliferative response of lymphocytes [in French]. Jpn J Med Sci Biol 1987;40:147151.

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  • 15. Seiki K, Sakabe K, Kawashima I, et al. Hormone and immune response, with special reference to steroid hormone 1. A short review. Tokai J Exp Clin Med 1990;15:191199.

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  • 16. Su S, Fang F, Liu Y, et al. The compensatory expression of reproductive hormone receptors in the thymus of the male rat following active immunization against GnRH. Gen Comp Endocrinol 2013;185:5766.

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Luteinizing hormone receptor expression by nonneoplastic and neoplastic canine lymphocytes

Alyssa M. Ettinger1Department of Animal and Rangeland Sciences, College of Agricultural Sciences, Oregon State University, Corvallis, OR 97331.

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Sabrina K. Gust1Department of Animal and Rangeland Sciences, College of Agricultural Sciences, Oregon State University, Corvallis, OR 97331.

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Michelle A. Kutzler1Department of Animal and Rangeland Sciences, College of Agricultural Sciences, Oregon State University, Corvallis, OR 97331.

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Abstract

OBJECTIVE

To investigate luteinizing hormone (LH) receptor expression in canine nonneoplastic and neoplastic lymph nodes, circulating nonneoplastic lymphocytes, and T-cell lymphoma (TCL) cell lines.

SAMPLE

Formalin-fixed, paraffin-embedded lymph nodes (5 neoplastic and 3 nonneoplastic) from 6 dogs, circulating lymphocytes from venous blood specimens obtained from 12 healthy dogs, and 3 TCL cell lines derived from 3 dogs with primary lymphoma.

PROCEDURES

Lymph node specimens were immunohistochemically stained for determination of LH receptor expression. Circulating nonneoplastic lymphocytes and TCL cell lines were evaluated for LH receptor expression by use of flow cytometry; circulating lymphocytes were also immunophenotyped. The mean percentage of cells positive for LH receptors was determined for each type of specimen. For the healthy dogs, percentages of circulating B and T lymphocytes that expressed LH receptors were assessed on the basis of sex and reproductive status.

RESULTS

The mean percentage of LH receptor-positive cells in canine neoplastic and nonneoplastic lymph nodes was 12.4% and 4.1%, respectively. For the healthy dogs, the mean percentage of circulating LH receptor-positive T lymphocytes was significantly higher in gonadectomized dogs (16.6%) than in sexually intact dogs (10.5%); the percentages of circulating LH receptor-positive B lymphocytes did not significantly differ by reproductive status. Among the 3 canine TCL cell lines, LH receptor expression ranged from 10% to 45%.

CONCLUSIONS AND CLINICAL RELEVANCE

In this study, LH receptor expression by canine neoplastic and nonneoplastic lymphocytes was detected. Research into the effects of downregulation of LH receptor activation in dogs with lymphoma is warranted.

Abstract

OBJECTIVE

To investigate luteinizing hormone (LH) receptor expression in canine nonneoplastic and neoplastic lymph nodes, circulating nonneoplastic lymphocytes, and T-cell lymphoma (TCL) cell lines.

SAMPLE

Formalin-fixed, paraffin-embedded lymph nodes (5 neoplastic and 3 nonneoplastic) from 6 dogs, circulating lymphocytes from venous blood specimens obtained from 12 healthy dogs, and 3 TCL cell lines derived from 3 dogs with primary lymphoma.

PROCEDURES

Lymph node specimens were immunohistochemically stained for determination of LH receptor expression. Circulating nonneoplastic lymphocytes and TCL cell lines were evaluated for LH receptor expression by use of flow cytometry; circulating lymphocytes were also immunophenotyped. The mean percentage of cells positive for LH receptors was determined for each type of specimen. For the healthy dogs, percentages of circulating B and T lymphocytes that expressed LH receptors were assessed on the basis of sex and reproductive status.

RESULTS

The mean percentage of LH receptor-positive cells in canine neoplastic and nonneoplastic lymph nodes was 12.4% and 4.1%, respectively. For the healthy dogs, the mean percentage of circulating LH receptor-positive T lymphocytes was significantly higher in gonadectomized dogs (16.6%) than in sexually intact dogs (10.5%); the percentages of circulating LH receptor-positive B lymphocytes did not significantly differ by reproductive status. Among the 3 canine TCL cell lines, LH receptor expression ranged from 10% to 45%.

CONCLUSIONS AND CLINICAL RELEVANCE

In this study, LH receptor expression by canine neoplastic and nonneoplastic lymphocytes was detected. Research into the effects of downregulation of LH receptor activation in dogs with lymphoma is warranted.

Lymphoma is the most common malignancy of hematopoietic origin in dogs, accounting for up to 24% of all canine cancers.1,2 The clinical signs and treatment options for dogs with lymphoma differ depending on whether the lymphoma is derived from B or T cells.3 Cells of B- and T-cell lymphomas express the unique cell surface proteins of B-cell lymphocytes (CD79a,4 CD20,4 PAX5,4 and CD215) and T-cell lymphocytes (CD3).4,5 Survival time for dogs with lymphoma is likely dependent on multiple factors, such as tumor grade and immunophenotype, breed and size of dog, and treatment type. For instance, survival times in dogs with lymphoma are reported to be longer in small than in large breeds.6 The prognosis for dogs with T-cell lymphoma is typically worse than that for dogs with B-cell lymphoma; the mean survival time for dogs with large-cell multicentric variants of T-cell lymphoma is 183 days, whereas the corresponding value for dogs with large-cell multicentric variants of B-cell lymphoma is 365 days.7

Factors such as age, breed, reproductive status, and age at the time of gonadectomy are all reported to be associated with the risk of lymphoma and lymphoma type.8 Lymphoma is most commonly detected in dogs 6 to 8 years of age.8 Although lymphoma can affect both purebred and mixed-breed dogs, several breeds, including Boxer, Bullmastiff, Basset Hound, Saint Bernard, Bulldog, German Shepherd Dog, Golden Retriever, Labrador Retriever, and Rottweiler, are reported to be at higher risk.4 In addition, Boxers appear to be at increased risk of developing T-cell lymphomas, whereas breeds such Basset Hound and Cocker Spaniel are more commonly affected by B-cell lymphomas.9 Studies10,11 of Vizslas and Golden Retrievers have revealed that gonadectomy is associated with an increased risk of lymphoma in those breeds.

To the authors' knowledge, a possible mechanism of action underlying an increased risk of lymphoma in gonadectomized versus sexually intact dogs has not been established. Negative feedback from the gonads to the adenohypophysis is lost following gonadectomy, resulting in supraphysiologic concentrations of circulating LH.12 The effect of persistently elevated LH concentrations in dogs was believed to be unimportant until LH receptors were discovered in nonneoplastic tissues outside the reproductive system (eg, urinary bladder, skin, and thyroid gland)a and in neoplastic tissues (eg, hemangiosarcoma and mastocytoma).b,c Luteinizing hormone receptor expression has also been detected on human circulating lymphocytes and in rodent lymphoid tissue.13–16 However, to the authors' knowledge, there are no published reports of LH receptor expression in canine lymphatic tissue. The purpose of the study reported here was to investigate LH receptor expression in canine nonneoplastic and neoplastic lymph nodes, circulating nonneoplastic lymphocytes, and T-cell lymphoma cell lines.

Materials and Methods

Sources of canine specimens

The Oregon State University Veterinary Diagnostic Laboratory provided 8 formalin-fixed, paraffin-embedded blocks of canine lymph node specimens (5 neoplastic and 3 nonneoplastic specimens from 6 dogs) that had been submitted between 2012 and 2017. Histologic evaluation of all lymph node specimens had been performed by board-certified veterinary pathologists at the veterinary diagnostic laboratory; a diagnosis of lymphoma was made for the 5 dogs with neoplastic lymph nodes, although information regarding the immunophenotype (T-cell or B-cell) was not available. The age of the dogs ranged from 6 to 12 years. Neoplastic specimens were obtained from 3 spayed females (1 Cocker Spaniel, 1 mixed-breed dog, and 1 dog of unknown breed), 1 sexually intact female (Bernese Mountain Dog), and 1 neutered male (mixed-breed dog); 3 nonneoplastic specimens were obtained from 1 neutered male German Shepherd Dog. Testicular tissue was obtained from a separate adult dog that had undergone routine castration for use as a positive control during immunohistochemical analysis of lymph node specimens.

Twelve healthy dogs were recruited from a local kennel club to provide blood samples from which circulating nonneoplastic lymphocytes were isolated. Informed consent was provided by all dog owners before samples were collected, and all procedures were approved by the Oregon State University Institutional Animal Care and Use Committee (protocol No. 4792). Among the 12 healthy dogs, there were 3 spayed females, 3 sexually intact females, 3 neutered males, and 3 sexually intact males. The mean age of the dogs was 6.7 years (range, 1 to 12 years); breeds included Brittany (n = 9) and Beagle, Chesapeake Bay Retriever, and German Shorthaired Pointer (1 each).

Three cryopreserved immortalized T-cell lymphoma cell lines (CLC, CLK, and EMA) derived from 3 adult female (reproductive status unknown) dogs (1 French Bulldog, 1 English Springer Spaniel, and 1 Great Pyrenees; age range, 6 to 8 years) with primary lymphoma were provided by the Laboratory of Veterinary Internal Medicine, Department of Veterinary Medicine, Yamaguchi University, Yamaguchi, Japan, for use in establishing cell cultures. The cells were shipped to our laboratory on dry ice.

Immunohistochemical analysis of lymph node specimens

Serial 6-μm-thick sections were cut from each tissue block and placed onto positively charged slides; 3 slides were prepared for each specimen, including 1 for use as a negative control and 1 that was stained with H&E stain only (for histologic examination). All sections were deparaffinized with xylene, rehydrated with graded ethanol solutions (100%, 75%, and 50%), and subjected to heat-induced epitope retrieval.d

The following procedures were performed for all slides, except those that were prepared for H&E staining only. Endogenous peroxidase activity was inactivated with 3% hydrogen peroxide, and nonspecific binding was blocked with 1% horse serume before application of a 1:50 dilution of goat polyclonal anti-human LH receptor (primary) antibody.f Negative controls for each tissue specimen were treated in a similar manner except that the primary antibody was not applied. Biotinylated horse anti-goat IgG (secondary) antibody was applied at a 1:10 dilution; slides were then incubated with preformed avidin-biotin-peroxidase complexe followed by horseradish peroxidase-based substrate.g Slides were counterstained with hematoxylin, dehydrated, and then mounted. The percentage of cells positive for LH receptors in each of 4 fields/slide was used to calculate the mean percentage of LH receptor-positive cells per slide; all slides were examined by 1 investigator (AME) at 400X magnification.

Isolation of circulating nonneoplastic lymphocytes

For each of the 12 healthy dogs, 6 mL of blood was collected into blood collection tubes containing EDTA by use of standard aseptic jugular venipuncture technique. Within 90 minutes after collection, each blood sample was sterilely transferred into a 15-mL conical centrifuge tube; an equal volume of PBSS-BSA-SA was added to each tube, and the tube contents were mixed by inversion. Six milliters of each blood sample mixture was then slowly layered on top of 4 mL of centrifugation mediumh (density, 1.077 g/mL) in a 15-mL centrifuge tube (ie, 12 separate tubes); the tubes were centrifuged at 400 × g for 30 minutes at room temperature (approx 23°C). Cells from the plasma-separation medium interface from each sample were collected by use of a plastic transfer pipette and placed into new 15-mL centrifuge tubes with up to 10 mL of PBSS-BSA-SA; the tubes were then centrifuged at 400 × g for 10 minutes at room temperature. After discarding the supernatant, the cell pellet from each sample was transferred to an Eppendorf tube containing up to 1 mL of PBSS-BSA-SA at 4°C, then centrifuged at 400 × g for 10 minutes at room temperature. The resulting pellets (circulating lymphocytes) were each resuspended in 700 μL of PBSS-BSA-SA at 4°C (resulting concentration, 130,000 to 200,000 lymphocytes/mL).

Flow cytometry

Circulating nonneoplastic lymphocytes—Six Eppendorf tubes containing isolated circulating lymphocytes were prepared for each dog; nonspecific binding by the cells was blocked by adding blocking serumi (1:10 dilution) to each tube and then incubating the samples on ice for 10 minutes. Ten microliters of goat polyclonal anti-human LH receptor (primary) antibodyf (1:10 dilution) was added to tubes 1 to 3 to identify LH receptors on lymphocytes; 10 μL of fluorescein isothiocyanate-conjugated mouse anti-dog CD3 antibodyj (1:10 dilution) and 10 μL of fluorochrome-conjugated mouse anti-dog CD21 antibodyk (1:10 dilution) were also added to tubes 2 and 3, respectively. To identify the percentage of T and B lymphocytes, 10 μL of fluorescein isothiocyanate-conjugated mouse anti-dog CD3 antibodyj (1:10 dilution) and 10 μL of fluorochrome-conjugated mouse anti-dog CD21 antibodyk (1:10 dilution) were added to tubes 4 and 5, respectively. Tube 6 was prepared with no primary antibody (negative control). After incubation at room temperature for 20 minutes, the lymphocytes were washed twice with PBSS-BSA-SA at 4°C to remove unbound primary antibody; 100 μL of PBSS-BSA-SA at 4°C was then added to each tube, and the contents were mixed by pipetting action. Ten microliters of a phycoerythrin-conjugated secondary antibody (donkey anti-goat IgGl; 1:10 dilution) was added to tubes 1, 2, 3, and 6 for each of the 12 healthy dogs. All tubes (n = 72) were then held on ice for 20 minutes. Each sample was washed twice with PBSS-BSA-SA to remove unbound antibodies, 200 μL of PBSS-BSA-SA at 4°C was added to each sample, and the tube contents were mixed by pipetting action. Pipette tip strainersm were used to transfer 200 μL of sample from each tube into a separate well of a 96-well plate (samples run in singlicate). The samples were analyzed at various wavelengths (ie, 375, 405, 488, 561, 638, and 808 nm) by use of a flow cytometer.n The data were analyzed by use of flow cytometry software.° Single cell staining was used to perform compensation, and fluorescence-minus-one controls were used to determine where gates should be set for LH receptor-positive cells.17

Cultured T-cell lymphoma cell lines—The thawed cell lines were each cultured separately in R10 complete mediump supplemented with 10% fetal bovine serum substituteq and penicillin (100 U/mL) with streptomycin (100 μg/mL)r in a humidified 5% CO2 incubator at 37°C. Fresh R10 complete medium was added to cell cultures every 2 to 3 days at a ratio of 1:4 or 1:9. On reaching 75% to 85% confluence, the cells were washed to remove culture medium, counted with a hemocytometer, and transferred to Eppendorf tubes. Nonspecific binding by cells was blocked by the described procedures for the circulating nonneoplastic lymphocytes; a 1:10 dilution of the primary antibodyf was then added, and the cells were incubated at room temperature for 20 minutes. A negative control was prepared in a similar manner for each cell line except that the primary antibody was omitted. Cell washing and addition of secondary antibodyl were performed as described for the preparation of circulating nonneoplastic lymphocytes for flow cytometry. Each cell line and its negative control were analyzed in triplicate (analysis of 40,000 to 50,000 lymphocytes/well) on a flow cytometer,n and the data were analyzed.° Single cell staining was used to perform compensation, and fluorescence-minus-one controls were used to determine where gates should be set for LH receptor-positive cells.17

Statistical analysis

Statistical analyses were performed with statistical software.s The Shapiro-Wilk test was used to evaluate the normality of the data on the mean percentage of LH receptor-positive cells per sample as obtained by immunohistochemical analysis and flow cytometry. The Student t test was used to compare the mean percentage of lymphocytes that expressed LH receptors in nonneoplastic versus neoplastic lymph node specimens. The Student t test was also used to compare the mean percentage of circulating nonneoplastic lymphocytes that expressed LH receptors in sexually intact versus gonadectomized dogs and in male versus female dogs. Significance was defined as a value of P < 0.05.

Results

Immunohistochemical analysis of lymph node tissue specimens revealed LH receptor expression by lymphocytes from neoplastic and nonneoplastic lymph nodes; no negative controls had positive results of staining (Figure 1). Luteinizing hormone receptors were expressed by a significantly (P = 0.02) higher percentage of lymphocytes from neoplastic than nonneoplastic lymph nodes (mean ± SD, 12.4 ± 5.4% and 4.1 ± 2.6%, respectively).

Figure 1—
Figure 1—

Representative photomicrographs of sections of archived neoplastic (A) and nonneoplastic (B) lymph node specimens from 2 dogs illustrating the relative expression of LH receptors, as determined immunohistochemically. Negative controls for each tissue specimen (upper right inset of each panel) were treated in a similar manner except that the primary antibody was not applied. Immunohistochemical LH receptor-specific stain with hematoxylin counterstain; bar = 10 μm for main and inset images.

Citation: American Journal of Veterinary Research 80, 6; 10.2460/ajvr.80.6.572

Luteinizing hormone receptors were expressed by circulating nonneoplastic B and T lymphocytes from all healthy dogs (range, 6.1% to 20.3% and 9.9% to 19.6%, respectively). Males and females did not differ significantly with regard to the percentage of circulating nonneoplastic B lymphocytes (mean ± SD, 19.7 ± 13.5% and 9.6 ± 5.4%, respectively; P = 0.06) and T lymphocytes (mean ± SD, 15.4 ± 8.1% and 11.8 ± 4.0%, respectively; P = 0.18) that expressed LH receptors (Figure 2). The percentage of circulating nonneoplastic T lymphocytes that expressed LH receptors was significantly (P = 0.049) higher in gonadectomized dogs than in sexually intact dogs (mean ± SD, 16.6 ± 7.8% vs 10.5 ± 2.3%, respectively); there was no corresponding significant (P = 0.45) difference with regard to LH receptor-positive circulating nonneoplastic B lymphocytes in gonadectomized and sexually intact dogs (mean ± SD, 14.2 ± 10.3% and 15.1 ± 12.8%, respectively).

Figure 2—
Figure 2—

Plots of the percentage of LH receptor (LHR)-positive circulating nonneoplastic T and B lymphocytes obtained from 12 healthy adult dogs, as determined by flow cytometry. Each circle represents the percentage of LHR-positive cells for 1 dog (6 dogs/subgroup). For each plot, the horizontal line in the middle represents the mean and the shorter horizonal lines at the top and bottom represent the upper and lower 95% confidence intervals for the mean. A—Data regarding T lymphocytes grouped on the basis of sex of the dogs. The percentage of LHR-positive T lymphocytes did not differ significantly (P = 0.18) between males and females. B—Data regarding B lymphocytes grouped on the basis of sex. The percentage of LHR-positive B lymphocytes did not differ significantly (P = 0.06) between males and females. C—Data regarding T lymphocytes grouped on the basis of reproductive status. The percentage of LHR-positive T lymphocytes in sexually intact dogs was significantly (P = 0.049) less than that in gonadectomized dogs. D—Data regarding B lymphocytes grouped on the basis of reproductive status. The percentage of LHR-positive B lymphocytes did not differ significantly (P = 0.45) between sexually intact and gonadectomized dogs.

Citation: American Journal of Veterinary Research 80, 6; 10.2460/ajvr.80.6.572

In each cell line, the cells that expressed LH receptors were smaller (on the basis of forward scatter data) and more granular (on the basis of side scatter data) than the cells that did not express LH receptors (Figure 3). The intensity of fluorescence of T-cell lymphoma cells that did and did not express LH receptors was plotted. The percentage of lymphocytes that expressed LH receptors in the 3 T-cell lymphoma cell lines varied (range, 10% to 45%); no negative controls had positive results of staining.

Figure 3—
Figure 3—

Representative scatterplot (A) and histogram (B) for the flow cytometric detection of LHR expression by T-cell lymphoma cells from an immortalized cell line derived from a dog with primary lymphoma. A—Forward (FSC) versus side scatter (SSC) plot indicating cell size and granularity, respectively, of T-cell lymphoma cells that did (green) or did not (blue) express LHR (P1 represents the percentage of lymphocytes within the gate). B—Histogram indicating the intensity of fluorescence of T-cell lymphoma cells that did (green peak) or did not (blue peak) express LHRs (P2 represents the percentage of cells that expressed LHRs, and the horizontal bar represents the range of fluorescent intensities of those cells).

Citation: American Journal of Veterinary Research 80, 6; 10.2460/ajvr.80.6.572

Discussion

In the present study, LH receptors were identified in several canine specimens: nonneoplastic and neoplastic lymph nodes, circulating nonneoplastic lymphocytes, and cultured T-cell lymphoma cells. To the authors' knowledge, this was the first published report to indicate that LH receptors are expressed in canine lymphatic tissue. Luteinizing hormone receptors have been identified in rodent lymphoid tissue and on human circulating lymphocytes.13,14,16 In the present study, LH receptors were expressed by a significantly higher percentage of lymphocytes from neoplastic (12.4%) than nonneoplastic (4.1%) lymph nodes. There was evidence of a similar difference in LH receptor expression between cultured T-cell lymphoma cells (10% to 45%) and circulating nonneoplastic T (9.9°% to 19.6°%) and B lymphocytes (6.1°% to 20.3%), although the samples sizes were small.

Male dogs had higher percentages of circulating nonneoplastic B and T lymphocytes with LH receptor expression, compared with female dogs. However, the apparent sex-related differences were not significant for the B- or T-lymphocyte data. The significantly higher percentage of circulating T lymphocytes with LH receptor expression in gonadectomized dogs, compared with that in sexually intact dogs, was an unexpected finding.

Further research is needed to evaluate the biological effects of any increases in lymphocyte-associated LH receptor expression that may occur in dogs following gonadectomy. Results of previous studies10,11 indicate that gonadectomy in dogs may be associated with increased risk of development of certain neoplasms. Research to investigate potential neoplasia-promoting effects of LH receptor activation (eg, cell proliferation, migration, or invasion) in lymphocytes is warranted. The identification of potential neoplasia-promoting effects of LH receptor activation in lymphocytes could lend support for additional studies of the usefulness of treatments that downregulate LH receptor expression in dogs with lymphoma.

ABBREVIATIONS

LH

Luteinizing hormone

PBSS-BSA-SA

PBS solution with 1% bovine serum albumin and 0.05% sodium azide

Acknowledgments

Supported in part by the E. R. Jackman Friends and Alumni and the Continuing Researchers Program (both from the College of Agricultural Sciences and Small Grants Program), Research Office, Oregon State University.

The authors declare that there were no conflicts of interest.

Presented in part as an abstract at the 2017 Society for Theriogenology Annual Conference, Fort Collins, Colo, August 2017; 6th Annual Symposium on the Alliance for Contraception in Cats and Dogs, Boston, Mass, July 2018; and 2018 Society for Theriogenology Annual Conference, Milwaukee, Wis, August 2018.

The authors thank Allison Ehrlich for assistance with developing the flow cytometry protocol and data analysis; Khawla Zwida for assistance with the immunohistochemical analyses; and Drs. Robert Bildfell, Barry Cooper, Jerry Heidel, Beth Valentine, and Christiane Löhr for performing the original histologic examinations of the lymph node specimens that were archived and later provided for use in this study.

Footnotes

a.

Ettinger A, Zwida K, Kutzler M. Normal and neoplastic canine lymphocytes express luteinizing hormone receptors (abstr). Clin Theriogenol 2017;9:428.

b.

Zwida K, Kutzler MA. Canine hemangiosarcoma expresses luteinizing hormone (LH) receptors (abstr). J Anim Sci 2016;94:201–202.

c.

Moccia V. Expression of luteinizing hormone receptor in canine cutaneous mast cell tumors. Graduate Thesis, Dipartimento di Medicina Veterinaria, Universitá degli studi di Bari Aldo Moro, Bari, Italy, 2018.

d.

Target retrieval solution, ready-to-use S1700, Dako, Carpinteria, Calif.

e.

Vectastain Elite ABC kit (peroxidase, horse anti-goat IgG) PK-6105, Vector Laboratories Inc, Burlingame, Calif.

f.

Goat polyclonal anti-human LHR antibody SC-26341, Santa Cruz Biotechnology Inc, Dallas, Tex.

g.

Vector NovaRED peroxidase (HRP) substrate kit SK-4800, Vector Laboratories Inc, Burlingame, Calif.

h.

Histopaque-1077, Sigma-Aldrich Corp, St Louis, Mo.

i.

Mouse seroblock FcR (FCR4G8) BUF041A, Bio-Rad Laboratories Inc, Hercules, Calif.

j.

Mouse anti-dog CD3 antibody MCA1774F, Bio-Rad Laboratories Inc, Hercules, Calif.

k.

Alexa fluor 647 mouse anti-canine CD21 antibody MCA1781A647, Bio-Rad Laboratories Inc, Richmond, Calif.

l.

Donkey anti-goat IgG PE 31860, ThermoFisher Scientific, Rockford, Ill.

m.

Bel-Art Flowmi 70-micron cell strainers for 1,000-μL pipette tips H13680-0070, SP Scienceware, Warminster, Pa.

m.

CytoFLEX, Beckman Coulter, Indianapolis, Ind.

o.

CytExpert software for CytoFLEX, Beckman Coulter, Brea, Calif.

p.

RPMI 1640 with glutathione 02-0105-0500, VWR Life Science, Visalia, Calif.

q.

Fetal Bovine Essence 3100, VWR Life Science, Randor, Pa.

r.

Penicillin, streptomycin, and glutamine solution SV3008201, GE Healthcare-HyClone Laboratories, South Logan, Utah.

s.

GraphPad Prism, version 8, GraphPad Software Inc, La Jolla, Calif.

References

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Contributor Notes

Address correspondence to Dr. Kutzler (michelle.kutzler@oregonstate.edu).