Dynamic alteration of neutrophil chemoattractant receptors regulates neutrophil chemotaxis in humans in vivo

Dynamic alteration of neutrophil chemoattractant receptors regulates neutrophil chemotaxis in humans in vivo

Vol. 191, No. 4S, October 2000 Surgical Forum Abstracts S7 Resuscitation induced pulmonary apoptosis and ICAM-1 expression are attenuated by the us...

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Vol. 191, No. 4S, October 2000

Surgical Forum Abstracts

S7

Resuscitation induced pulmonary apoptosis and ICAM-1 expression are attenuated by the use of ketone Ringer’s solution in rats Hasan Alam, MD, Brenda Austin BS, Elena Koustova PhD, Peter Rhee, MD, FACS. Uniformed Services University of the Health Sciences, 4301 Jones Bridge Road, Bethesda, MD, 20814, USA. Phone (301) 295-9833, e-mail: [email protected] Introduction: Resuscitation with Lactated Ringer’s (LR) solution in rats has been shown to cause immune mediated lung injury as measured by upregulation of apoptosis and increased expression of intracellular adhesion molecule-1 (ICAM-1). We hypothesized that the use of energy substrates such as ketone bodies (ß-hydroxybutyrate) would protect against this injury. Methods: Anesthetized Sprague Dawley rats were subjected to a bleed of 27ml/kg over 10 minutes. Over the next 75 minutes of shock, another 8ml/kg of blood was withdrawn at a steady rate. Following randomization, resuscitation fluids were infused over 45 minutes during which a simultaneous bleed of 8ml/kg was performed. The animals were sacrificed one hour later. Western blot technique was used for the determination of pro-apoptotic protein (bax), anti-apoptotic protein (bcl-2), apoptotic fragments of poly ADP-ribose polymerase (PARP) and the expression of ICAM-1. Lung sections were also subjected to immunostaining. Results: Lactated Ringer’s (LR) resuscitation caused a significant increase in pulmonary apoptosis and ICAM-1 expression compared to sham. Animals resuscitated with Ketone Ringer’s and plasma did not show this upregulation. APOPTOSIS

Fluid Groups

n

Sham NR LR (3:1 volume) KR (3:1 volume) Plasma (1:1 volume)

6 7 8 7 7

Bax/Bcl-2 Ratio

PARP Cleavage

ICAM-1 EXPRESSION Bax Immunostaining

0.84 ⫾ 0.36 0% 13.30 ⫾ 1.14 2.17 ⫾ 0.59 0% 23.88 ⫾ 2.52 2.58 ⫾ 0.48* 50%‡ 27.64 ⫾ 4.36* 1.48 ⫾ 0.47 28.6% 11.42 ⫾ 1.45 0.95 ⫾ 0.19 28.6% 18.22 ⫾ 3.73

Western Blot (OD)

Immunostaining

0.65 ⫾ 0.03 0.78 ⫾ 0.02 0.79 ⫾ 0.01* 0.66 ⫾ 0.03 0.75 ⫾ 0.02

33.8 ⫾ 6.0 55.8 ⫾ 4.4 70.3 ⫾ 7.0* 38.6 ⫾ 6.2 51.7 ⫾ 6.3

Data are group mean ⫾ SEM. Immunostaining data presented as positive cells/mm2. * p ⬍ 0.05 using ANOVA/ Dunnets test for multiple comparison against sham group. ‡ p ⬍ 0.05 using chi-square test between the LR group and sham. NR ⫽ no resuscitation; KR ⫽ Ketone Ringer’s resuscitation; OD ⫽ optical density.

Conclusions: Substitution of lactate with ß-hydroxybutyrate in the resuscitation fluid offers protection against resuscitation induced pulmonary apoptosis and ICAM-1 upregulation.

had a transient increase in TNFRII surface expression at 6 hours but not TNFRI. Addition of the transcriptional inhibitor actinomycin D abrogated the effect of CpG ODN on soluble TNFRI and II and mRNA. Anti-IL10 and anti-TNF antibody did not change the results. Conclusions: De novo, non-IL10 and non-TNF dependent TNF receptor transcription, translation and shedding is an additional potential counterinflammatory effect of bacterial DNA; interactions between CpG and other bacterial products (e.g. LPS) remain to be studied.

Intra-abdominal sepsis attenuates inflammation mediated increases in microvascular permeability at remote sites Lorenzo Ferri MD, Jose Pascual MD, Andrew Seely MD, Nicolas V Christou MD PhD, FACS. Division of General Surgery, McGill University. N.V. Christou MD, Royal Victoria Hospital, 687 Pine Ave W., room C5.53, Montreal, Qc., Canada H3A 1A1. Tel (514) 8431531 Fax (514) 843-1430 Introduction: We have previously demonstrated that intra-abdominal sepsis decreases leukocyte delivery to remote sites. Given that leukocytes have been implicated in the pathogenesis of endothelial injury in sepsis leading to a diffuse vascular leakage, we hypothesized that this decrease in leukocyte delivery to remote sites in sepsis actually translates into decreased vascular leakage at these sites, thus challenging the current dogma. Methods: CD1 male mice were randomized to a generalized sepsis group by cecal ligation and puncture (CLP); a local infection group (orchitis-ORC); CLP ⫹ ORC; and control. The cremaster muscle was prepared for intravital microscopy 24 hours post procedure and FITC labeled albumin was injected intravenously. Leukocyte rolling and adherence were counted off-line. Post capillary venule leakage was determined by the permeability index (perivenular/intravenular fluorescence). Statistical significance was determined using ANOVA (p ⬍ 0.05).

Macrophage TNF-␣ receptor synthesis, expression and shedding in response to CpG oligonucleotide exposure Daniel P Raymond, M.D., Long Jin M.D., Shawn J Pelletier M.D., Traves D Crabtree M.D., Thomas G Gleason M.D., Timothy G Pruett M.D. FACS, Robert G Sawyer M.D., FACS. (Contact-Raymond) University of Virginia, Dept. of Surgery; Bldg. MR4, Rm. 3150; Lane Rd.; Charlottesville, VA 22908, USA Phone: (804) 924-5329 FAX: (804) 924-1218 Introduction: The immunomodulatory role of unmethylated cytosineguanine sequences (CpG) in bacterial DNA has been well documented. We have previously demonstrated that pre-exposure of macrophages to synthesized CpG-containing 18-mers (ODN) causes an IL-10 dependent suppression in TNF-␣ production in response to LPS challenge. Since TNF-␣ and TNF-␣ receptor (TNFRI and II) expression are tightly regulated responses, we hypothesized that TNFRI and II shedding would also be affected by CpG.

Results: CLP did not increase leukocyte rolling, adherence, or vascular permeability of cremaster venules (ns), rather CLP ⫹ ORC decreased local infection (ORC) mediated increases in adherence and vascular leakage (p ⬍ 0.01). Conclusions: Intra-abdominal sepsis does not increase vascular permeability systemically at 24 hours; rather, intra-abdominal sepsis significantly attenuates inflammation induced increases in vascular leakage at remote sites. The decreased leukocyte rolling and adherence at remote sites in sepsis represent a likely mechanism for this effect.

Methods: RAW 264.7 cells were incubated for various time periods with CpG-ODN or Non-CpG containing 18-mers (NonCpG-ODN). Supernatants were assayed for soluble TNFRI and II by ELISA, cell surface expression of TNFRI and II by FACS and TNFRI and II mRNA by Ribonuclease Protection Assay.

Dynamic alteration of neutrophil chemoattractant receptors regulates neutrophil chemotaxis in humans in vivo

Results: Exposure to CpG, but not NonCpG-ODN, resulted in a significant, time-dependent increase in TNFRI and TNFRII production (figure 1), after a rise in TNFR-mRNA (figure 2). Cells treated with CpG

Andrew JE Seely MD, Jean-Francois Naud, Giuseppina Campisi B Sc, Jose L Pascual MD, Lorenzo Ferri MD, Nicolas V Christou MD PhD, FRCS(C). Division of General Surgery, McGill University,

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Surgical Forum Abstracts

Montreal, Quebec. Correspondence: Nicolas V. Christou, MD, PhD, FRCS(C), Professor of Surgery, Royal Victoria Hospital, 687 Pine Ave. W., Room C5-53, Montreal, Quebec, Canada H3A 1A1; Phone: 514-843-1531; Fax: 514-843-1430. Introduction: In vivo regulation of polymorphonuclear neutrophil (PMN) chemoattractant receptors and their functional significance remain poorly understood. We hypothesized that (1) alteration in receptors for both PMN specific (e.g. Interleukin-8) & non-specific (e.g. C5a) chemoattractants occurs in humans in vivo, and (2) this regulates the ability of PMN’s to physically respond to chemoattractants. Methods: We evaluated chemoattractant receptor expression (C5a receptor (C5aR), Interleukin-8 RA (IL-8RA), & B (IL-8RB) with immunofluorescent monoclonal antibodies & flow cytometry) and in vitro chemotaxis to C5a & IL-8 (Boyden chambers), in three functionally separate groups of human PMN: (1) circulating PMN (venipuncture), (2) exudate PMN (collected with skin blister skin window method), both from healthy controls (n ⫽ 10), and (3) circulating PMN from patients with sepsis (n ⫽ 6, APACHE II mean 26.6, range 15–38). Data presented as mean ⫾ SD; ANOVA and paired t-test used for analysis. Results: Compared to circulating control PMN, septic PMN had a reduction in C5aR and chemotaxis to C5a; whereas exudate PMN had an elevation in C5aR, but no significant increase in chemotaxis to C5a (Figure 1). Conversely, exudate PMN displayed reduced IL-8RA, IL8RB, & chemotaxis to IL-8; and septic PMN displayed reduced IL-8RA & IL-8RB, and no change in chemotaxis to IL-8 (Figure 2).

J Am Coll Surg

P38 was assayed 30mins after BLP, LPS or LPS ⫹ BLP stimulation in tolerised and non-tolerised macrophages by western immunoblotting. Results: BLP tolerance resulted in 0% mortality compared to controls (60%: p ⬍ 0.05). BLP tolerance also protected against LPS mortality, 30% vs 80% (control): p ⬍ 0.05 and BLP ⫹ LPS lethality, 0% vs 60% (control): p ⬍ 0.05. Although LPS tolerance protects against its own lethality, it did not protect against BLP or BLP ⫹ LPS mortality. Pre-treatment of macrophages with BLP significantly attenuated LPS, BLP and LPS ⫹ BLP stimulation-induced TNF-␣ production and supressed the p38 dependent signal transduction pathway. Conclusions: BLP tolerance protects against its own lethal effects as well as endotoxin. This effect appears to be mediated through supression of p38 and TNF-␣.

Cardiothoracic Surgery I: Innovations/Neonatal-Congenital Laser versus mechanical transmyocardial revascularization: do you need a light? Keith A Horvath, MD, Noam Belkind, Rodney Greene, BS, Bonnie Kane, David D McPherson, MD, David A Fullerton, MD. Feinberg Cardiovascular Research Institute, Northwestern Univ. Med. School, 251 E. Chicago Avenue, Wesley 1030, Chicago, IL 60611, USA, (312) 908-3121 Introduction: The success of transmyocardial laser revascularization in treating patients with severe coronary artery disease has lead to a renewed effort to achieve the same benefit by mechanical means. While both methods create a similar transmural channel the importance of laser light in creating these channels is unknown. The purpose of this study was to determine whether mechanical revascularization improves myocardial function and increases angiogenesis as compared to laser revascularization in chronically ischemic myocardium.

Conclusions: Simultaneous evaluation of chemoattractant receptors & chemotaxis in three distinct groups of human PMN demonstrated: (1) PMN chemoattractant receptor expression is capable of dynamic alteration in vivo; (2) selective reduction of chemoattractant receptors provides a mechanism to explain reduced PMN delivery in septic patients; and (3) changes in PMN chemotaxis correlate with corresponding changes in chemoattractant receptors, but receptor alteration does not necessarily alter chemotaxis. These results suggest that change in receptor expression is necessary, but not sufficient to alter PMN chemotaxis.

Bacterial lipoprotein induces endotoxin (LPS) independent tolerance to systemic inflammatory response syndrome Majella Doyle AFRCSI, Jiang Huai Wang PhD, H. Paul Redmond FRCSI. Department of Academic Surgery, Cork University Hospital Wilton, Cork, Ireland. Tel. 35321901290 fax. 35321344230. Introduction: Sublethal doses of endotoxin (LPS) induce tolerance through a p38-dependent kinase pathway and protect against the lethal effects of a further LPS challenge. Tolerance is characterised by a diminished production of tumour necrosis factor-␣ (TNF-␣). The role of bacterial lipoprotein (BLP), an ubiquitous bacterial cell wall component, in tolerance induction has not been investigated. We hypothesised that BLP, could induce tolerance against itself and endotoxin and that this tolerance is characterised by reduced p38 kinase activity and TNF-␣ production. Methods: Male MF-1 mice were randomised into three groups (n ⫽ 30/group). Group 1 received a single BLP tolerant dose (IP, 10mg/kg). Group 2 received LPS (IP, 10mg/kg) tolerance. Following induction of tolerance, all mice were challenged with high doses of LPS (IP, 45mg/ kg), BLP (IP, 45mg/kg) or a combination of LPS ⫹ BLP (IP, 30 ⫹ 30mg/kg). Group 3 received only high doses of the above agents. Mortality was assessed over 48hrs. TNF-␣ was assayed from peritoneal macrophage culture supernatants, 6hrs after BLP, LPS or LPS ⫹ BLP stimulation ⫾ 24hr pre-treatment with BLP or medium, by ELISA.

Methods: In a model of chronic ischemia by Ameroid occlusion of the circumflex artery, 18 domestic pigs were randomized to: Group I treatment with revascularization by an excimer laser, Group II - mechanically revascularization using the same fiber that delivers the excimer laser light, or Group III - no treatment. Segmental contraction was assessed at rest and with dobutamine stress echocardiography. Six weeks after revascularization the animals were restudied at rest and with stress. Angiogenesis was assessed by counting blood vessels identified by factor VIII immunohistochemistry. Results: Compared to the mechanical group, transmyocardial revascularization by an excimer laser completely restored resting function in chronically ischemic myocardium. Control animals did not demonstrate significant recovery of function.

Laser Mechanical

Baseline Resting Function

Postop Resting Function

p value

0.38 ⫾ 0.07cm 0.49 ⫾ 0.08cm

0.67 ⫾ 0.11cm 0.53 ⫾ 0.05cm

⬍0.05 ns

Laser treatment of the ischemic zone led to an increase in the number of blood vessels per 200⫻ high powered field vs. mechanical treatment. Blood Vessels/hpf Ischemic Non-ischemic Zone Zone Laser Mechanical

31 ⫾ 2 17 ⫾ 3

16 ⫾ 2 15 ⫾ 3

p value ⬍0.05 ns

Control animals demonstrated no increase in blood vessels/hpf.

Conclusions: The addition of laser light to transmyocardial revascularization leads to improvement in function and increased angiogenesis in chronically ischemic myocardium. These data indicate that laser revascularization is more effective than transmyocardial revascularization by mechanical means.