. 2015 Apr 7;19(1):149.

doi: 10.1186/s13054-015-0883-z.

Human severe sepsis cytokine mixture increases β2-integrin-dependent polymorphonuclear leukocyte adhesion to cerebral microvascular endothelial cells in vitro

Chris Blom¹, Brittany L Deller², Douglas D Fraser^{3

4

5

6

7}, Eric K Patterson⁸, Claudio M Martin^{9

10}, Bryan Young¹¹, Patricia C Liaw^{12

13}, Payam Yazdan-Ashoori¹⁴, Angelica Ortiz¹⁵, Brian Webb¹⁶, Greg Kilmer¹⁷, David E Carter¹⁸, Gediminas Cepinskas^{19

20}

Affiliations

¹ Department of Physiology and Pharmacology, Western University, 1151 Richmond Str. North, London, ON, N6A 5C1, Canada. christopher.blom@mail.utoronto.ca.
² Department of Physiology and Pharmacology, Western University, 1151 Richmond Str. North, London, ON, N6A 5C1, Canada. bdeller2@uwo.ca.
³ Department of Physiology and Pharmacology, Western University, 1151 Richmond Str. North, London, ON, N6A 5C1, Canada. douglas.fraser@lhsc.on.ca.
⁴ Children's Health Research Institute, 800 Commissioners Road East, London, ON, N6C 2V5, Canada. douglas.fraser@lhsc.on.ca.
⁵ Centre for Critical Illness Research, Lawson Health Research Institute, 800 Commissioners Rd East, London, ON, N6C 6B5, Canada. douglas.fraser@lhsc.on.ca.
⁶ Department of Paediatrics, Western University, 100 Collip Circle, London, ON, N6G 4X8, Canada. douglas.fraser@lhsc.on.ca.
⁷ Department of Clinical Neurological Sciences, Western University, 339 Windermere Road, London, ON, N6A 5A5, Canada. douglas.fraser@lhsc.on.ca.
⁸ Centre for Critical Illness Research, Lawson Health Research Institute, 800 Commissioners Rd East, London, ON, N6C 6B5, Canada. eric.patterson@lhsc.on.ca.
⁹ Centre for Critical Illness Research, Lawson Health Research Institute, 800 Commissioners Rd East, London, ON, N6C 6B5, Canada. cmartin1@uwo.ca.
¹⁰ Department of Medicine, Western University, 1151 Richmond Str. North, London, ON, N6A 3K6, Canada. cmartin1@uwo.ca.
¹¹ Department of Clinical Neurological Sciences, Western University, 339 Windermere Road, London, ON, N6A 5A5, Canada. bryan.young@lhsc.on.ca.
¹² Department of Medicine, McMaster University, 1280 Main Street West, Hamilton, ON, L8S 4K1, Canada. patricia.liaw@taari.ca.
¹³ The Thrombosis and Atherosclerosis Research Institute, 237 Barton Str. East, Hamilton, ON, L8L 2X2, Canada. patricia.liaw@taari.ca.
¹⁴ Department of Physiology and Pharmacology, Western University, 1151 Richmond Str. North, London, ON, N6A 5C1, Canada. pashoori@gmail.com.
¹⁵ Children's Health Research Institute, 800 Commissioners Road East, London, ON, N6C 2V5, Canada. angelica@angelicaortiz.ca.
¹⁶ Thermo Fisher Scientific, 3747 N Meridian Rd, Rockford, IL, 61105, USA. brian.webb@thermofisher.com.
¹⁷ Thermo Fisher Scientific, 3747 N Meridian Rd, Rockford, IL, 61105, USA. greg.kilmer@thermofisher.com.
¹⁸ London Regional Genomics Centre, Robarts Research Institute, 1151 Richmond Str. North, London, ON, N6A 5B7, Canada. dcarter@robarts.ca.
¹⁹ Centre for Critical Illness Research, Lawson Health Research Institute, 800 Commissioners Rd East, London, ON, N6C 6B5, Canada. gcepinsk@uwo.ca.
²⁰ Department of Medical Biophysics, Western University, 1151 Richmond Str. North, London, ON, N6A 5C1, Canada. gcepinsk@uwo.ca.

PMID: 25882865
PMCID: PMC4409718
DOI: 10.1186/s13054-015-0883-z

Human severe sepsis cytokine mixture increases β2-integrin-dependent polymorphonuclear leukocyte adhesion to cerebral microvascular endothelial cells in vitro

Chris Blom et al. Crit Care. 2015.

. 2015 Apr 7;19(1):149.

doi: 10.1186/s13054-015-0883-z.

Authors

Affiliations

¹ Department of Physiology and Pharmacology, Western University, 1151 Richmond Str. North, London, ON, N6A 5C1, Canada. christopher.blom@mail.utoronto.ca.
² Department of Physiology and Pharmacology, Western University, 1151 Richmond Str. North, London, ON, N6A 5C1, Canada. bdeller2@uwo.ca.
³ Department of Physiology and Pharmacology, Western University, 1151 Richmond Str. North, London, ON, N6A 5C1, Canada. douglas.fraser@lhsc.on.ca.
⁴ Children's Health Research Institute, 800 Commissioners Road East, London, ON, N6C 2V5, Canada. douglas.fraser@lhsc.on.ca.
⁵ Centre for Critical Illness Research, Lawson Health Research Institute, 800 Commissioners Rd East, London, ON, N6C 6B5, Canada. douglas.fraser@lhsc.on.ca.
⁶ Department of Paediatrics, Western University, 100 Collip Circle, London, ON, N6G 4X8, Canada. douglas.fraser@lhsc.on.ca.
⁷ Department of Clinical Neurological Sciences, Western University, 339 Windermere Road, London, ON, N6A 5A5, Canada. douglas.fraser@lhsc.on.ca.
⁸ Centre for Critical Illness Research, Lawson Health Research Institute, 800 Commissioners Rd East, London, ON, N6C 6B5, Canada. eric.patterson@lhsc.on.ca.
⁹ Centre for Critical Illness Research, Lawson Health Research Institute, 800 Commissioners Rd East, London, ON, N6C 6B5, Canada. cmartin1@uwo.ca.
¹⁰ Department of Medicine, Western University, 1151 Richmond Str. North, London, ON, N6A 3K6, Canada. cmartin1@uwo.ca.
¹¹ Department of Clinical Neurological Sciences, Western University, 339 Windermere Road, London, ON, N6A 5A5, Canada. bryan.young@lhsc.on.ca.
¹² Department of Medicine, McMaster University, 1280 Main Street West, Hamilton, ON, L8S 4K1, Canada. patricia.liaw@taari.ca.
¹³ The Thrombosis and Atherosclerosis Research Institute, 237 Barton Str. East, Hamilton, ON, L8L 2X2, Canada. patricia.liaw@taari.ca.
¹⁴ Department of Physiology and Pharmacology, Western University, 1151 Richmond Str. North, London, ON, N6A 5C1, Canada. pashoori@gmail.com.
¹⁵ Children's Health Research Institute, 800 Commissioners Road East, London, ON, N6C 2V5, Canada. angelica@angelicaortiz.ca.
¹⁶ Thermo Fisher Scientific, 3747 N Meridian Rd, Rockford, IL, 61105, USA. brian.webb@thermofisher.com.
¹⁷ Thermo Fisher Scientific, 3747 N Meridian Rd, Rockford, IL, 61105, USA. greg.kilmer@thermofisher.com.
¹⁸ London Regional Genomics Centre, Robarts Research Institute, 1151 Richmond Str. North, London, ON, N6A 5B7, Canada. dcarter@robarts.ca.
¹⁹ Centre for Critical Illness Research, Lawson Health Research Institute, 800 Commissioners Rd East, London, ON, N6C 6B5, Canada. gcepinsk@uwo.ca.
²⁰ Department of Medical Biophysics, Western University, 1151 Richmond Str. North, London, ON, N6A 5C1, Canada. gcepinsk@uwo.ca.

PMID: 25882865
PMCID: PMC4409718
DOI: 10.1186/s13054-015-0883-z

Abstract

Introduction: Sepsis-associated encephalopathy (SAE) is a state of acute brain dysfunction in response to a systemic infection. We propose that systemic inflammation during sepsis causes increased adhesion of leukocytes to the brain microvasculature, resulting in blood-brain barrier dysfunction. Thus, our objectives were to measure inflammatory analytes in plasma of severe sepsis patients to create an experimental cytokine mixture (CM), and to use this CM to investigate the activation and interactions of polymorphonuclear leukocytes (PMN) and human cerebrovascular endothelial cells (hCMEC/D3) in vitro.

Methods: The concentrations of 41 inflammatory analytes were quantified in plasma obtained from 20 severe sepsis patients and 20 age- and sex-matched healthy controls employing an antibody microarray. Two CMs were prepared to mimic severe sepsis (SSCM) and control (CCM), and these CMs were then used for PMN and hCMEC/D3 stimulation in vitro. PMN adhesion to hCMEC/D3 was assessed under conditions of flow (shear stress 0.7 dyn/cm(2)).

Results: Eight inflammatory analytes elevated in plasma obtained from severe sepsis patients were used to prepare SSCM and CCM. Stimulation of PMN with SSCM led to a marked increase in PMN adhesion to hCMEC/D3, as compared to CCM. PMN adhesion was abolished with neutralizing antibodies to either β2 (CD18), αL/β2 (CD11α/CD18; LFA-1) or αM/β2 (CD11β/CD18; Mac-1) integrins. In addition, immune-neutralization of the endothelial (hCMEC/D3) cell adhesion molecule, ICAM-1 (CD54) also suppressed PMN adhesion.

Conclusions: Human SSCM up-regulates PMN pro-adhesive phenotype and promotes PMN adhesion to cerebrovascular endothelial cells through a β2-integrin-ICAM-1-dependent mechanism. PMN adhesion to the brain microvasculature may contribute to SAE.

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Figures

**Figure 1**
**PMN adhesion to hCMEC/D3 following stimulation with severe sepsis cytokine mixture (SSCM).** hCMEC/D3 were grown on laminar flow microchannels and interacted with PMN following stimulation of hCMEC/D3, PMN, or both PMN and hCMEC/D3, with CCM or SSCM. In these experiments, hCMEC/D3 were stimulated with CCM or SSCM for five hours. PMN were stimulated for 10 minutes immediately before interacting them with hCMEC/D3. PMN adhesion to hCMEC/D3 was assessed in the presence of flow (laminar shear stress 0.7 dyn/cm²). n = 7; *P <0.05 as compared to corresponding control cytokine mixture (CCM). CCM-control cytokine mixture; SSCM-severe sepsis cytokine mixture; hCMEC/D3-human cerebral microvascular endothelial cells/D3; PMN-polymorphonuclear leukocytes.

**Figure 2**
**Effects of anti-β** ₂ **-integrin (CD18) antibody on severe sepsis cytokine mixture (SSCM)-induced PMN adhesion to hCMEC/D3.** PMN were first stimulated with CCM or SSCM for 10 minutes and subsequently treated with anti-β₂-integrin (CD18) function neutralizing antibody (or control isotype antibody) for an additional 15 minutes. PMN adhesion to stimulated PMN **(A)** or stimulated PMN and hCMEC/D3 **(B)** was assessed in the presence of flow (laminar shear stress 0.7 dyn/cm²). n = 4. *P <0.05 as compared to corresponding control cytokine mixture (CCM); # P <0.05 as compared to isotype IgG treatment. CCM-control cytokine mixture; SSCM-severe sepsis cytokine mixture; hCMEC/D3-human cerebral microvascular endothelial cells/D3; PMN-polymorphonuclear leukocytes; IgG-immunoglobulin G; CD18-cluster of differentiation 18.

**Figure 3**
**Effects of anti-LFA-1 and anti-Mac-1 antibodies on severe sepsis cytokine mixture (SSCM)-induced PMN adhesion to hCMEC/D3.** PMN were first stimulated with CCM or SSCM for 10 minutes and subsequently treated with anti-LFA-1 or anti-Mac-1 function neutralizing antibody (or control isotype antibody) for an additional 15 minutes. PMN adhesion to stimulated PMN **(A)** or stimulated PMN and hCMEC/D3 **(B)** was assessed in the presence of flow (laminar shear stress 0.7 dyn/cm²). n = 4. *P <0.05 as compared to corresponding control cytokine mixture (CCM); # P <0.05 as compared to isotype IgG treatment. CCM-control cytokine mixture; SSCM-severe sepsis cytokine mixture; hCMEC/D3-human cerebral microvascular endothelial cells/D3; PMN-polymorphonuclear leukocytes; IgG-immunoglobulin G; LFA-1 - lymphocyte function-associated antigen 1; Mac-1 - macrophage-1 antigen.

**Figure 4**
**Effects of anti-ICAM-1 antibody on severe sepsis cytokine mixture (SSCM)-induced PMN adhesion to hCMEC/D3.** hCMEC/D3 were stimulated with CCM or SSCM for 5 hours, treated with anti-ICAM-1 function neutralizing antibody for 30 minutes, and interacted with CCM- or SSCM-stimulated (10 minutes) PMN to assess PMN adhesion in the presence of flow (laminar shear stress 0.7 dyn/cm²). n = 6; *P <0.05 as compared to corresponding control cytokine mixture (CCM); # P <0.05 as compared to isotype IgG treatment. CCM-control cytokine mixture; SSCM-severe sepsis cytokine mixture; hCMEC/D3-human cerebral microvascular endothelial cells/D3; PMN-polymorphonuclear leukocytes; Ab-antibody; ICAM-1 – intercellular adhesion molecule-1.

**Figure 5**
**Effects of severe sepsis cytokine mixture (SSCM) on adhesion molecule ICAM-1 and VCAM-1 expression in hCMEC/D3.** hCMEC/D3 were stimulated for 4 hours with either CCM or SSCM preceding analysis of *VCAM-1* and *ICAM-1* gene expression using qPCR. Data is presented as gene expression normalized to *GAPDH* and *18S* expression, relative to hCMEC/D3 treated with serum-free VascuLife EnGS-Mv cell culture media (n = 5). CCM-control cytokine mixture; SSCM-severe sepsis cytokine mixture; hCMEC/D3-human cerebral microvascular endothelial cells/D3; ICAM-1 – intercellular adhesion molecule-1; VCAM-1 – vascular cell adhesion molecule-1; 18S-18S ribosomal RNA; GAPDH-glyceraldehyde 3-phosphate dehydrogenase; qPCR- quantitative polymerase chain reaction.

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References

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Human severe sepsis cytokine mixture increases β2-integrin-dependent polymorphonuclear leukocyte adhesion to cerebral microvascular endothelial cells in vitro

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Human severe sepsis cytokine mixture increases β2-integrin-dependent polymorphonuclear leukocyte adhesion to cerebral microvascular endothelial cells in vitro

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