The Antibody for IL-8 Required Frozen Tissue Which Was Cut Into 20 Μm Sections and Fixed

EXPANDED METHODS HYPERTENSION/2004/029322R2

NEUTROPHILS INFILTRATE RESISTANCE SIZED VESSELS OF SUBCUTANEOUS

FAT IN WOMEN WITH PREECLAMPSIA

Courtney E. Leik, Ph.D. and Scott W. Walsh, Ph.D.

Departments of Obstetrics and Gynecology, and Physiology

Virginia Commonwealth University Medical Center

Richmond, Virginia

Address for correspondence: Scott W. Walsh, Ph.D., Department of Obstetrics and

Gynecology, P.O. Box 980034, Virginia Commonwealth University Medical Center,

Richmond, VA 23298-0034. Tel (804) 828-8468, Fax (804) 828-0573, e-mail [email protected]

1 EXPANDED METHODS HYPERTENSION/2004/029322R2

METHODS

Study Subjects

Subcutaneous fat biopsies were collected from patients at MCV Hospitals of

Virginia Commonwealth University Medical Center. Fat biopsies were collected at the time of cesarean section from normal pregnant (NP) patients (n = 6) and preeclamptic

(PE) patients (n = 5) or at the time of abdominal or minimally invasive surgery from normal, non-pregnant (NNP) patients (n = 4). Surgeries for normal non-pregnant women were performed for removal of uterine myomas or for tissue biopsies. The criteria for normal pregnancy were a maternal blood pressure of less than 140/90 mmHg, no proteinuria, and no other complications. Preeclampsia was defined as hypertension greater than 140/90 mmHg on at least two separate measurements six hours apart and proteinuria greater than 300 mg/24 hours or 2 plus dipstick. Women in active labor were excluded from the study. The criteria for normal, non-pregnant patients were a blood pressure of less than 140/90 mmHg, and no systemic inflammatory conditions, such as diabetes. All patients were non-smokers. Patients were matched for body mass index

(BMI). Informed consent was obtained prior to surgery. This study was approved by the

Virginia Commonwealth University Office of Research Subjects Protection. Procedures were in accordance with institutional guidelines.

Collection of Fat

Subcutaneous fat biopsies were used because subcutaneous fat is a highly vascularized tissue representative of the systemic vasculature and it is easily obtained at the time of surgery. Fat biopsy pieces (approximately 1 cm x 1 cm x 1 cm) collected

2 EXPANDED METHODS HYPERTENSION/2004/029322R2 from each patient were divided and placed immediately in either 10% neutral buffered formalin or snap-frozen in liquid nitrogen and stored at –70C until tissue processing.

Immunohistochemistry

Vessels of subcutaneous fat were stained for IL-8, ICAM-1 and CD66b, a granulocyte-specific antigen. CD66b is up-regulated and secreted upon granulocyte activation. CD66b staining primarily represents neutrophils because they comprise 96% of the granulocyte population.

The antibody for IL-8 required frozen tissue which was cut into 20 µm sections and fixed in acetone (-20C). Formalin-fixed tissue was used for ICAM-1 and CD66b staining. Tissue was paraffin-embedded and cut in 8 µm sections. Antigen retrieval was performed with microwave citrate pretreatment for ICAM-1 and CD66b.

Immunohistochemical staining was performed using VECTASTAIN Elite ABC

(Universal) and DAB Substrate Kits (Vector Laboratories, Burlingame, CA) according to manufacturer’s instructions, except working solutions for ICAM-1 and CD66b were diluted in 100mM phosphate buffer, pH=7.5, without 0.9% NaCl. Slides were processed with a ST 5050 automated staining machine (Vision Instruments, Australia). Antibodies used were: 1) mouse IgG anti-human monoclonal antibody specific for IL-8 (1:50,

BioSource International, Camarillo, CA); 2) mouse IgG anti-human monoclonal antibody against ICAM-1 (CD54, 1:100, Zymed Laboratories, San Francisco, CA); and 3) mouse

IgM anti-human monoclonal antibody specific for CD66b (1:500, BD BioSciences, San

Diego, CA). Mouse IgG monoclonal isotype standard (pre-diluted, Zymed Laboratories,

San Francisco, CA) or mouse IgM monoclonal isotype standard (1:500, BD

BioSciences, San Diego, CA) were used as negative controls. The titer of the

3 EXPANDED METHODS HYPERTENSION/2004/029322R2 biotinylated antibody was increased from 1:50 to 1:25 for CD66b to improve cross- reactivity with the primary IgM antibody. To identify endothelium and vascular smooth muscle, sequential sections were immunostained with a rabbit anti-human monoclonal antibody directed at Factor VIII (1:600 for frozen tissue and 1:400 for formalin-fixed tissue, Zymed Laboratories, San Francisco, CA) or a mouse anti-human monoclonal antibody directed at -smooth muscle actin (1:6000 for frozen tissue and 1:3000 for formalin-fixed tissue, Sigma, St. Louis, MO), respectively. DAB reagent resulted in brown staining of antigens. Tissue was counterstained with alcian blue and methyl green.

Data Analysis

Lumen width was measured for each vessel and vessels between 10 µm and

200 µm, which represent resistance-sized vessels, were analyzed for intensity of staining by an investigator blinded to the identity of the tissue. Average vessels scored per patient (±SE) were: IL-8: NNP = 24 ± 13 (n = 2), NP = 37 ± 9 (n = 4) and PE = 26 ±

5 (n = 4); ICAM-1: NNP = 75 ± 6 (n = 4), NP = 156 ± 47 (n = 5) and PE = 72 ± 18 (n =

5); CD66b: NNP = 134 ± 40 (n = 4), NP = 204 ± 20 (n = 6) and PE = 128 ± 45 (n = 5).

Fewer vessels were scored per patient for IL-8 because the frozen sections used for IL-

8 were smaller than the paraffin-embedded sections used for ICAM-1 and CD66b.

Vessel staining was graded using a visual score ranging from zero to four (absent to intense staining). For CD66b, the score included neutrophil involvement in the vessel.

Staining scores were verified by optical density (OD) measurements using image analysis software (Image Pro Plus, Media Cybernetics, Silver Spring, MD). Sections with CD66b staining were also evaluated for: 1) percent of vessels with staining for

4 EXPANDED METHODS HYPERTENSION/2004/029322R2 neutrophils; 2) percent of vessels with neutrophils flattened and adhered to endothelial cells; 3) percent of vessels with neutrophils infiltrated into the intimal space; and 4) average number of neutrophils per vessel.

Statistical Analysis

One-way analysis of variance with Newman-Keuls post-hoc test was used to analyze continuous data. Kruskal-Wallis with Dunn’s post hoc test was used for score data and continuous data if variances were not equal. Regression analysis was done to correlate staining scores with OD. Power of analyses were greater than 0.9, = 0.05

(two-tailed). A statistical software application was used (Prism, GraphPad Software Inc.,

San Diego, CA).