Visualization of immunoreactive growth hormone in cultured peripheral bovine lymphocytes

Growth Hormone & IGF Research 22 (2012) 59–63

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Visualization of immunoreactive growth hormone in cultured peripheral bovine lymphocytes Sabine Klein, Nahid Parvizi ⁎ Department of Functional Genomics and Bioregulation, Institute of Farm Animal Genetics Mariensee, FLI, Höltystr. 10, 31535 Neustadt, Germany

a r t i c l e

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Article history: Received 3 August 2011 received in revised form 10 January 2012 accepted 20 January 2012 Available online 15 February 2012 Keywords: Growth hormone Intracellular localization Con-focal microscopy Lymphocyte

a b s t r a c t Growth hormone (GH) has been shown to be released by immune cells in vitro. Thus, the intracellular confinement of GH immunoreactivity was investigated in cultured bovine lymphocytes using con-focal microscopy. Peripheral blood lymphocytes from cows in early pregnancy (10–20 days post insemination; pi) or during mid-pregnancy (day 110–140 pi) were harvested and cultured for 48 h in presence of phytohemagglutinin-M (PHA-M) or served as controls. Thereafter, immunocytochemistry was conducted using a homologous GHantibody. Double staining (GH-antibody and directly DYE 549 labeled CD3-antibody) was performed to classify the cells. Con-focal laser scanning was applied verifying the immunofluorescence labeling. Interestingly, the presence of GH immunoreactivity in the cytoplasm, which indicates GH synthesis, was restricted to small cells. Whereas, few T-like cells revealed surface bound GH. Lowest immunoreactivity, concerning the number of the total labeled cells as well as the intensity of labeling was recorded in early pregnancy. Stimulation with PHA-M enhanced total labeled cells in early pregnancy. In contrast, PHA-M had no such effects in midpregnancy. The results confirm the specific regulation of synthesis of lymphocytic GH during pregnancy in the cow. The identification of cells producing GH and the elucidation of the mechanisms underlying the expression of GH in larger number of cells during mid-pregnancy than in the early pregnancy need further investigations. © 2012 Elsevier Ltd. All rights reserved.

1. Introduction Bidirectional communication between the immune and the neuroendocrine system is mediated by a common set of signaling molecules including hormones, cytokines, and receptors of these mediators [1–3]. Growth hormone (GH) is one of the first classical hormones, which was identified in lymphocytes [4]. Lymphocytes not only produce GH [5–8] but also express GH receptor [5–9]. The lymphocytic GH is apparently similar to its pituitary counterpart in the human [10] and in the pig [8]. Growth hormone is involved in the development and regulation of immune system [11,12]. It modulates the migration of developing T cells [13] and improves the leukocyte's function in ovariectomized old rats [14]. Further, GH enhances the function of thymus in HIV-1-infected men [15]. Chung et al. [16] reported a negative regulation of calcium binding pro-inflammatory S100 proteins by GH in human white blood cells. At gene level, a close relationship is found between GH-gene expression and the activity of the hemolytic complements (CH50 and AH50) in the pig [17]. Taken all this together there is a close relationship between immune system and GH.

⁎ Corresponding author at: Department of Functional Genomics and Bioregulation, Institute of Farm Animal Genetics, FLI, Höltystrasse 10, Mariensee, D-31535, Neustadt a. Rbg., Germany. Fax: +49 5034 871 247. E-mail address: nahid.parvizi@fli.bund.de (N. Parvizi). 1096-6374/$ – see front matter © 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.ghir.2012.01.001

We previously demonstrated adrenocorticotropin (ACTH) synthesis in bovine lymphocytes, which is highlighted during the pregnancy [18]. We also have shown that bovine lymphocytes harvested from cyclic as well as pregnant cows secret GH when cultured in vitro [8,19]. Thus, the present study aimed to specify to which extent the cellular immunoreactive GH can be visualized in cultured bovine lymphocytes. To achieve this aim, lymphocytes from cows in early (10 to 20 days post insemination; pi) and mid-pregnancy (110 to 140 days pi) were cultured with or without phytohemagglutinin-M (PHA-M). The types and number of GH-labeled lymphocytes, as well as the intensity and distribution of labeling within the cells were analyzed. 2. Materials and methods 2.1. Preparation of lymphocytes A total of 12 pregnant Holstein Frisian cows (HF, 5.4 ± 1.2 years old; in their 1–3 lactation) from the research farm of the Institute of Farm Animal Genetics were included in the experiment. Animals were divided into two groups according to the stage of pregnancy. Group one was composed of four cows during early pregnancy (10 to 20 days pi). Group two was comprised of 8 cows in midpregnancy (110–140 days pi). The peripheral lymphocytes were separated as described previously [18]. A minimum of 250 ml blood was

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Fig. 1. Bovine lymphocytes labeled with anti-bovine GH raised in rabbits and visualized with anti-rabbit antibody conjugated to FITC: A) The FITC channel of a control sample without primary GH antibody. The inset shows the projection overlay of differential interference contrast and fluorescence channel of this region. For more detail see the text. B) FITC-channel projection showing two GH positive lymphocytes (*). The two GH-labeled cells are connected via a tread-like structure (→), which is also GH positive and could indicate cell to cell communication. The inset demonstrates the arrangement of different lymphocytes as overlay with the differential interference contrast. C) Projection overlay of a region with small cells with GH immunoreactivity (green) attached to a larger cluster of T cells identified by CD3 immunoreactivity (red). This arrangement was characteristic for lymphocytes stimulated with PHA-M. The lack of yellow color suggests the absence of GH in these CD3 cells. D) A 2-channel projection overlay of a sample with one small GH positive cell (green arrow) between 3 large T cells. Two of the T cells are positive for the CD3 label (red arrows). All scales 5 μm. All cells are PHA-M stimulated.

withdrawn from the jugular vein between 8 and 9 o' clock a.m. The EDTA mixed blood was centrifuged. Thereafter, the buffy coat was re-suspended in Hanks balanced salt solution (HBSS, H2387, SIGMA, Taufkirchen, Germany). Lymphocytes were separated with lymphodex (H9L5049, Inno-Train, Kronberg, Germany), transferred to fresh tubes and washed 3 times with HBSS. Remaining erythrocytes were lysed for 10 s in distilled water. Cells (1 × 10 6 per ml) were transferred to the culture medium consisting of 1:1 (v:v) RPMI-1640 (SIGMA, Germany) and HBSS. This culture medium was supplemented with 1% normal calf serum (NCS, N4762, SIGMA) and 1% antibiotic and antimycotic solution (A5655, Sigma). The cells were seeded in four-well plates (Nunc Brand Products, Darmstadt, Germany) and incubated at 37 °C and in 5% CO2 and 95% air. After 24 h of adaptation, medium was changed for all cultures and 25 μg/ml PHA-M (from Phaseolus vulgaricus L8902, SIGMA) was added to half of the cell cultures. After 48 h of incubation cells were washed once in PBS, and fixed in 3.7% formaldehyde (1.04002, Merck Darmstadt, Germany) for 15 min and dehydrated in ascending concentrations of methanol (30%, 50% and 100%) solution. Samples of 30 × 10 6cells were stored at −20 C for immunocytochemistry. A CD3 antibody was used to verify T cells (see Immunocytochemistry). Viability of the cells was assessed in fresh cultures and after the incubation period using trypan blue exclusion test. This test revealed seeding of more than 96% living cells in fresh cultures. After three days of culture, the proportion of living cells in controls varied between 82 and 94%, whereas in PHA-M stimulated cultures 74 to 85% of cells proved alive.

2.2. Immunocytochemistry Samples of 30 × 10 6cells each were rehydrated in 50, 70 and 100% PBS, and washed once in 0.02 M PBS. To block nonspecific binding, the rehydrated cells were incubated in PBS containing 5% normal goat serum (NGS) (DAKO, X0907, Glostrup, Denmark), 2% bovine serum albumin, and 0.2% Triton X100 for 30 min. Thereafter, the cells were transferred to a PBS buffer containing anti-bovine growth hormone antibody raised in rabbit (AbD Serotec, 4750–0956, diluted 1:4000, Morphosys, Oxford, UK,) or the anti-human CD3 antibody raised in rabbit (SIGMA C 7930, diluted 1:800), 0.2% triton X100, 0.1% sodium azide (SIGMA) and 1% NGS. After overnight incubation at 4 °C, the cells were washed 3 times in PBS and incubated with goat anti rabbit IgG conjugated to FITC (1:800, FI-1000, Vector, Burlingham, USA) for 90 min. The cells were mounted onto silanized slides and covered with vectashield H-100 (Axxora, Loerach, Germany) sealed with a cover slip and nail varnish. The specificity of the binding of the GH antibody was determined by a 3 h pre-incubation of the antibody solution with 10 μg/ml bovine GH (H070/H, Batch B980173, BioGenesis, New Fields, UK,) and 0.5% BSA before the application on the cells. Furthermore, in all immunocytochemical setups, the primary antibody was omitted in one sample of PHA-M-stimulated lymphocytes to estimate the nonspecific binding of the secondary antibody. The GH specific fluorescent signal was completely abolished, when the primary antibody was omitted (Fig. 1A). Moreover, preincubation of the GH-antibody with bovine GH for 3 h abolished the

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Fig. 2. Intracellular immunoreactivity of GH in two small cells (A) and membrane associated labeling of a large (T-like) lymphocyte (B). One single optical section at the position of the blue lines within the optical stack and orthogonal X*Z (top) and Y*Z (right) views of the total Z-stacks are shown for the positions of the green and orange lines respectively. The overlay with the DIC contrast is used to indicate the cellular morphology. A) The distribution of the GH peptide throughout the cytoplasm is demonstrated for the cell 1 (right upper corner) in the Y*Z view and for the cell 2 (left side, attached to three cells without GH labeling) in the X*Z view. B) Section through a cell with GH labeling restricted to the cell surface neighboring an unlabeled lymphocyte. Hence, the optical section is positioned iso-centrically the membrane-associated GH-immunoreactivity appears as a ring for all perspectives. Scales 5 μm.C–E) GH–FITC-labeled (green arrows) and subsequently directly CD3–Dy549 labeled (red arrow) bovine lymphocytes. C) A single optical section of 800 nm thickness positioned at the blue line within the optical stack as indicated in the orthogonal X*Z- and Y*Z-views. The orthogonal X*Z and Y*Z views are positioned at the green and orange line respectively. Small lymphocytes (green arrows) are shown embedded within larger CD3-labeled (red arrow) T lymphocytes. Only one cell (3, green arrow) is visualized in the X*Y optical section. The other cell (4, green arrow) is situated above this Z-level as demonstrated in the X*Z view and could only be seen in this orthogonal view. CD3-labeled T cells are attached to these small cells. D) The 3D-projection of the channel detecting the GH label for the region visualized in C. E) 3D projection of the CD3 label of the region shown in C. No co-localization between the GH- and CD3 is evident. Scales 5 μm. Cells shown in C to E are PHA-M stimulated.

positive intracellular GH-fluorescent signal as well as that bound to the cell membrane completely (Data not shown). Lymphocytes from one animal (day 132 of pregnancy) were double labeled with both the GH- and the CD3 primary antibodies. Following the labeling with the GH antibody the cells were subjected to a short blocking step with 1% newborn calf serum, 1% NGS and 1% rabbit serum. Thereafter, the cells were incubated with directly DY 549-labeled CD3-antibody at 4 °C overnight. After this incubation, the cells were washed 3 times and processed as described above. This double staining procedure enabled us to identify T cells (CD3+) in addition to the GH-labeled cells. 2.3. Microscopy A con-focal laser scanning system LSM510 connected to an Axioplan 200 (Carl Zeiss Micro Imaging GmbH, Jena, Germany) was applied quantifying the immunofluorescence intensities. The Argon laser (488 nm) was used to excite the FITC conjugated GH. The Helium–Neon green laser (543 nm) was used to excite directly

Dye 549-labeled CD3 in multi tracking mode. The detection of the gross cell morphology was realized by the transmission channel in differential interference contrast (DIC). All images had been taken at high resolution (63x Apochromat, NA. 1.4) and minimum pixel size was 50 × 50 × 800 nm for images of 24.8 × 24.8 × 20 μm. A series of 4 to 6 representative images was taken from each of animal in each stage of pregnancy. The analysis of positively labeled cells and intensity of labeling was performed by a person who was not aware of the identity of the samples. Background intensity was adjusted to 50 to 200 of a scale differentiating 4096 gray values for the channel detecting GH immunoreactivity. Specific GH labeling had been accepted above a measured intensity of 600 that is three times above the background. The signal to noise ratio had been calculated dividing the mean signal intensity by 200. 2.4. Statistics Statistical evaluation was performed employing a two-way ANOVA with two fixed factors: pregnancy stage as first parameter

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and PHA-M treatment as second parameter (Sigma Stat 3.5, Systat Software, Erkrath, Germany). Post-hoc pair wise multiple mean comparisons were performed according to Holm-Sidak's procedure. Results are presented as means ± SEM. Differences were considered significant at P ≤ 0.05. 3. Results and discussion 3.1. Cells labeled for GH High resolution con-focal imaging was used to differentiate between extracellular bound and the intracellular accumulated GH. Optical sections of 800 nm thicknesses were analyzed sequentially. This analysis confirmed the accumulation of GH-immunoreactivity predominantly in small lymphocytes (Figs. 1B&D and 2A) with rough surface. We used a microscopic field (max. 58 μm × 58 μm × 20 μm each) containing about 20 cells to analyze GH-labeled cells. Up to 3 GHcontaining cells were observed in a single field of the total lymphocytes. Noteworthy, some of these small GH-labeled cells were connected to each other by a tread- or bridge-like structure (Fig. 1B), which was also GH positive. At present, we have no explanation for the nature, function or source of these connections. Further the small GH-labeled cells are partially aggregated to numerous T-lymphocyte-like cells (Fig. 1C). Some of these T-lymphocytes carry GH immunoreactivity at their outer surface (Fig. 2B). Application of the GH- and the CD3 antibodies on the same cells indicated independent immunoreactivity of GH-labeled small cells and CD3-labeled T cells (Fig. 2C–D). Only one tenth of analyzed fields contained cells with GH immunoreactivity at their surface (Fig. 2B). The surface restricted GH immunoreactivity on cells most probably originates from extracellular GH bound to the cell surface. The electron microscopic atlas of rat lymphocytes [20] describes B-lymphocytes by their considerable smaller diameters than Tlymphocytes. Moreover, B-lymphocytes are characterized by a rough surface structure coursed by numerous pseudopodia differentiating them from the smooth surface of T-lymphocytes. This gross morphological appearance of the cell surface helps to distinguish between T-and B-type lymphocytes. In the present experiment, the GH-intracellular labeling appears concentrated in cells of small diameter and rich of pseudopodia, which may be defined as B-type lymphocytes. Hattori et al. [21] reported GH-gene expression in CD-19 human B-Lymphocytes. However, we did not apply any exclusive method to identify the B cells.

where a high degree of aggregation was observed, small cells (Figs. 1D and 2A) or small cell clusters (Figs. 1C and 2C) were located in proximity of the CD3T cells (Fig. 2C-D) suggesting a paracrine action of lymphocytic GH. Other investigators have also proposed that the action of lymphocytic GH and other hormones produced by immune cells is most probably mediated by para- and autocrine mechanisms [22,23]. The comparison between the number of GH immunoreactive lymphocytes and the release of GH by lymphocytes is speculative, nevertheless, the number of GH containing cells seems to be in contrast to the release of GH from lymphocytes in vitro. Lymphocytes harvested from cows during mid pregnancy (100–140 day) release lower amounts of GH than lymphocytes collected from cows in early pregnancy [19].The number of lymphocytes containing immunoreactive GH, however, is lower in early-pregnancy than during midpregnancy. This negative association may reflect a rapid release of the de novo synthesized GH in early pregnancy and, thus, a depletion of GH content in the cells. It is also feasible that the high GH release causes a down regulation of GH synthesis via an auto-or paracrine action. Storage of GH in the cells due to a delayed release in mid pregnancy can also not be ruled out. Stimulation of cells with PHA-M had no significant effect in mid-pregnancy. This observation is in

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GH immunoreactive cells

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b b

3

b 2

a 1

0

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10-20 days pi 7

Signal to noise ratio of GH-IR

3.2. Pregnancy-dependent changes in immunoreactive GH cells The number of GH-labeled lymphocytes in controls was significantly (P ≤ 0.05) larger during mid-pregnancy (2.09 ± 0.36 cells/ image) than during early pregnancy (0.86 ± 0.45). Stimulation with PHA-M caused an increase (P ≤ 0.01) in the number of GH positive cells in early pregnancy (2.74 ± 0.38 cells/image) but not during mid-pregnancy (2.65 ± 0.34 cells/image; Fig. 3A). The intensity of GH immunoreactivity, expressed as signal to noise ratio (SNR), was 3.80 ± 0.47 times above background in early pregnancy. The intensity increased to a SNR of 5.50 ± 0.29 in mid-pregnancy, which was significantly greater (P ≤ 0.05) than the SNR in early pregnancy (Fig. 3B). The PHA-M challenge did not significantly change the SNR (Fig. 3B). Dixit et al. [5] reported the presence of immunoreactive GH cells among the porcine lymphocytes. Weigent and Blalock [6,7] described B cells, macrophages and stimulated T-helper cells as the main blood cell types synthesizing GH in rats. Whereas, human B cells appear to be the only blood cells producing GH [21]. In the present study, GH immunoreactivity was predominantly seen within the cytoplasm of small cells confirming these cells as the main source of the lymphocytic GH production in the cow. In the stimulated lymphocytes,

A

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PHA-M

110-140 days pi

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b

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a 4 3 2 1 0 C

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Fig. 3. Quantification of GH immunoreactivity in bovine lymphocytes: A) Numbers of GH-positive small cells in each imaged field (20 cells). B) Fluorescence intensities as signal to noise ratio (SNR). Means and standard error of means are presented using 4 to 6 images from 7 (110–140 days pi) or 4 cows (10–20 days pi); Different letters indicate significant differences, in A) a vs. b P ≤ 0.01, in B) a vs. b P ≤ 0.05.

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agreement with earlier findings, which indicated that PHA-M did not change the GH release by lymphocytes obtained from HF cows during mid-pregnancy [24]. 4. Conclusions

[10]

[11] [12]

Visualization of immunoreactive GH demonstrated that GH synthesis seems to be restricted to bovine small blood cells. Whereas, few other cells (most probably T cells) carry GH on their surface. Identification of these cells and characterization of the immune cells producing GH need further studies. Further investigations are also essential to elucidate the mechanisms underlying the expression of GH in larger number of cells during mid-pregnancy than in the early pregnancy.

[13]

[14]

[15]

Acknowledgments

[16]

We thank R. Wittig and H.G. Sander for their excellent technical support.

[17]

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