Macrophage Inhibitory Factor (MIF) and High-Mobility Group-1 Protein (HMG-1) in Children Undergoing Cardiopulmonary Bypass

PURPOSE: To document the presence or absence of the proinflammatory cytokines macrophage migration inhibitory factor (MIF) and high-mobility group-1 protein (HMG-1) in the serum and myocardium of children undergoing cardiopulmonary bypass (CPB) and to correlate the presence or absence of these cytokines with clinical outcome after CPB

BACKGROUND: MIF, a 12.5 kD protein discovered in the 1960s as a substance produced by sensitized T lymphocytes involved in delayed type hypersensitivity, has emerged as a key cytokine in the innate immune response to infectious and inflammatory stimuli. Sources of MIF include monocytes/macrophages, the anterior pituitary, liver, kidney, spleen, and brain. MIF is released in response to various stimuli including lipopolysaccharide (LPS), toxic shock syndrome toxin-1, tumor necrosis factor (TNF), and interferon (INF). Once released, MIF promotes the secretion of other proinflammatory mediators by macrophages and T-cells thus intensifying the body's immune response. In addition, MIF has the unique ability to override the anti-inflammatory and immunosuppressive effects of glucocorticoids (Calandra et al., Nature Medicine, Feb 2000). MIF in combination with LPS potentiates lethality in murine endotoxemia models, and administration of anti-MIF antibodies increases survival in murine sepsis models. (Calandra et al., Nature Medicine, Feb 2000). Additionally, MIF knockout mouse models are resistant to lethal doses of LPS (Bozza et al., J Exp Med, Jan 1999).

HMG-1, a 30kD protein discovered in 1973, is a nonhistone chromatin-associated protein that serves as a DNA binding protein involved in nucleosome stabilization, facilitation of gene transcription, and as a modulator of steroid home receptor activity. More recently HMG-1 has been implicated as a monocyte/macrophage derived cytokine that serves as a late mediator of endotoxin lethality (Wang et al., Science 1999). Murine and human macrophages/monocytes release large amounts of HMG-1 18 hours after exposure to bacterial endotoxin. Serum HMG-1 levels rise 16-36 hours after LPS administration in murine lethal endotoxemia models. Administration of anti-HMG antibodies attenuates lethality in these models even when administered 2 hours after LPS exposure. Purified rHMG-1 is lethal to LPS-responsive and LPS-resistant mice. HMG-1 levels are increased in patients with sepsis and are higher in non-survivors than survivors. (Wang et al., Science 1999). HMG levels are also increased in patients with hemorrhagic shock (Ombrellino et al., Lancet 1999). HMG-1 induces TNF release by cultured human peripheral blood monocytes (Andersson et al., J Exp Med, 2000).

Cardiopulmonary bypass (CPB) triggers an inflammatory state associated with endotoxemia and cytokine elevation (Lequier et al., Chest, Jun 2000). Cytokines are known to have profound effects on cardiac function in sepsis. Cardiac depression occurs commonly after CPB and may be related to cytokine elevation. The role of MIF and HMG-1 in the CPB-induced inflammatory state are unknown. We have demonstrated the presence of MIF in murine heart tissue in response to LPS and are currently performing time course studies and functional assays examining murine cardiac myocyte response to MIF stimulus. In order to examine the clinical relevance of our laboratory data and to further characterize the inflammatory state occurring after CPB, we would like to obtain human data correlating MIF levels in human serum and myocardium with clinical outcome following CPB. Additionally, since the myocardial depression seen after CPB occurs 12-24 hours after CPB suggesting that a late mediator of inflammation may be important in post-CPB myocardial depression, we would like to assess for HMG-1 in the serum of patients undergoing CPB and correlate the presence or absence of HMG-1 with clinical outcome.

CONCISE SUMMARY OF PROJECT: We propose to study a population of infants and children undergoing operative repair of congenital heart disease during which there is an expectation that cardiac tissue will be removed. Patients will have an assessment of cardiac function by echocardiography as well as blood assays for tumor necrosis factor (TNF), interleukin-6, interleukin-8, interleukin-10, complement 3a, MIF, and HMG-1 prior to surgery. Cardiac tissue, removed as a planned part of the surgical procedure will be assayed for MIF as well as for evidence of apoptosis, a potential mechanism of cell death and myocardial dysfunction in response to MIF or HMG-1. The patients will be monitored in the PICU for a number of clinical outcome indicators including vital signs, fluid status, inotropic requirement, acid-base status, etc. Blood will be obtained prior to surgery and at 1, 8, 24, 48, and 72 hours post-operatively for cytokine assays to characterize the inflammatory response of each patient. We will then determine if MIF and/or HMG-1 levels correlate with clinical outcome.