27 July = 2005

29=20 July 2005

H5N1:=20 Hypercytokinemia and Hemophagocytosis?

What=20 is the mechanism of the high case fatality rate of influenza A virus = H5N1 in=20 humans both in the initial outbreak in 1997 in Hong Kong and in the = ongoing=20 outbreak that since 2004 has killed 55 of the 109 lab-confirmed cases in = Vietnam, Thailand, Cambodia, and Indonesia? Answers to this question could = influence=20 anti-H5N1 therapies, and potentially impact development of a safe and = effective=20 vaccine. =

One=20 hypothesis is that that the H5N1 virus stimulates much higher amounts of = proinflammatory cytokines including Tumor Necrosis Factor alpha = (TNF-alpha),=20 interleukin 6 (IL-6), and Interferon Induced Protein 10 (IP-10). In = severe cases=20 these cytokines trigger the reactive hemophagocytic syndrome (RHPS), = which in=20 other diseases is linked with multiorgan damage (e.g., to the bone = marrow,=20 liver, kidneys, and blood clotting system) and death. =

Hemophagocytosis=20 refers to the pathological process whereby human macrophage white blood = cells=20 ingest (=93phagocytose=94) other white blood cells (such as = neutrophils), red blood=20 cells, and platelets (that normally help blood to clot). A review of the = literature reveals that the =93reactive=94 form has been reported to = occur in=20 response to over 20 infectious diseases ranging from viruses (e.g., = EBV), to=20 bacteria (e.g., brucella), to fungi (e.g., histoplasmosis) to rickettsia = (e.g.,=20 Q-fever).

The=20 common denominator for the reactive hemophagocytic syndrome appears to = be marked=20 overproduction of proinflammatory cytokines such as TNF-alpha by=20 monocyte-macrophage white blood cells. The ideal therapy is to treat and = cure=20 the underlying cause, namely the inciting infectious disease. In = practice,=20 however, potent anti-inflammatory medications, such as corticosteroids, = are=20 sometimes given, particularly when the infection is not identified, a = specific=20 anti-microbial drug is unavailable, or the patient is very=20 ill.

Evidence=20 to support the H5N1 virus link to hypercytokinemia and hemophagocytic = syndrome=20 is limited due to the small number of patients with cytokine studies, = tissue=20 biopsies, and autopsies. In the initial 1997 H5N1 outbreak in humans in = Hong=20 Kong 6/18 symptomatic = patients died=20 and 2/6 had autopsies performed. Both showed striking reactive = hemophagocytic=20 syndrome (To KF et al. J Med Virol 2001;63:242-46). A third = patient had a=20 bone marrow aspirate that showed occasional hemophagocytic activity. In=20 retrospect, cytokine levels were measured on stored serum samples and = showed=20 increased TNF-alpha, IL-6, and soluble IL-2 receptor (Chan PKS. Clin=20 Infect Dis 2002;34=20 (suppl):558-564).

The=20 H5N1 virus from Hong Kong in 1997 was also shown to induce high levels = of the proinflammatory cytokines = TNF-alpha,=20 IL-6, and interferon-gamma when used to infect cultures of primary human = macrophages (Cheung CY et al. Lancet 2002;360:1831-37). However, this 1997 virus = appeared to be=20 protected against the antiviral effect of TNF-alpha, at least in part by = a=20 change in the Non-Structural (NS) gene that required a glutamic acid = residue at=20 position 92 in the NS1 molecule (Seo SH et al. Nature Med = 2002;8:950-954.=20 Lipatov AS et al. J Gen Virol 2005;86:1121-1130. Virus = Research=20 2004;103:107-113). = Overproduction=20 of TNF-alpha by laboratory-cultured macrophages infected with this 1997 = H5N1=20 virus could be blocked by inhibitors of =20 p38 MAPK (Mitogen Activated Protein Kinase) (Lee DC et al. J = Virol=20 2005;79:10147-54).

Similarly,=20 in 2003 two patients were diagnosed with H5N1 pneumonia after travel to = Fujian=20 province, China (Shinya K et al. J Virol 2005;79:9926-9932). = Their viruses were reported to = stimulate=20 TNF-alpha and IP-10 in human macrophage cultures (Guan Y et al. = PNAS=20 2004;101:8156-61). They = also had=20 elevated serum levels of TNF-alpha and IP-10, and monokine induced by = interferon=20 gamma (MIG) (Peiris JS et al. Lancet 2004; 363:582-83).=20

In=20 the current H5N1 outbreak of 2004-2005 serum cytokine levels of IP-10 = were=20 strikingly high (37,000pg/ml on day 5 of illness) in one 6 year old = child from=20 Thailand, although his autopsy did not show RHPS, possibly due to = treatment with=20 methylprednisolone (Uiprasertkul M et al. Influenza A H5N1 replication = sites in=20 humans. Emerg Infect Dis 2005; 11:1036-1041). A 2^nd = patient=20 who died had a limited exam of tissues and hemophagocytic activity was = seen in=20 the liver (Ungchusak K et al. N Engl J Med 2005;352:333-340). No = cytokine=20 data has been reported from this patient.

Continuing=20 research is needed to look for hypercytokinemia and stimulation of p38 = MAPK,=20 hemphagocytosis, and mutations in the virus=92 NS1 molecule as well as = the=20 hemagglutinin (HA) gene, to understand better how H5N1 causes its high = mortality=20 rate. Development of a safe and effective vaccine against H5N1, as well = as new=20 therapies based on inhibition of TNF-alpha and other cytokines, may = benefit from=20 such integrated research.

Daniel=20 R. Lucey, MD

Adjunct=20 Professor of Microbiology and Immunology

Georgetown=20 University School of Medicine

Director,=20 Centre for Biologic Counterterrorism and Emerging = Diseases

Department=20 of Emergency Medicine, Washington Hospital Center

Washington,=20 DC

e-mail:=20 Daniel.R.Lucey@Medstar.net=

Hong=20 Kong, SAR 29 July=20 2005