**8. Pathophysiologic and molecular mechanism of HLH**

Although significant progress in understanding the genetics and pathophysiology of primary HLH has been achieved during recent years, the pathogenesis of acquired forms of HLH is still not fully understood. An exaggerated immune response is the final common pathway of HLH, however, there are multiple roads leading to it (Arceci, 2008; Janka, 2009). The immune response is often triggered by different stimulants (e.g., infection) and the underlying inherited or acquired immune defect. It has been proposed that the clinical presentation of HLH is due to uncontrolled activation of immune cells, macrophages and CD8+ T lymphocytes (cytotoxic), leading to a massive release of various mediators of inflammation such as TNF-α (tumor necrosis factor α), interleukin(IL)-6, IL-8, IL-10, IL-12, IL-18, interferon γ, macrophage inflammatory protein (MIP 1-α), and hematopoietic growth factors (e.g., GM-CSF) (Filipovich, 2009; Henter et al., 1991a, 1996, 2007; Janka & Schneider, 2004; Osugi et al., 1997). IL-10 with its anti-inflammatory properties plays many important roles in the regulation of autoimmune inflammatory responses, particularly of systemic autoimmune disorders such as HLH/MAS. The role of IL-10 as part of an important regulatory mechanism involved in HLH has long been proposed (Behrens et al., 2011; Benveniste et al., 2000; Osugi et al., 1997). Recently, the roles of T regulatory cells in HLH have also been discussed (Verbsky & Grossman, 2006). Low or absent NK-cell function is present in many HLH patients and results in difficulties in termination of the exaggerated immune response (Filipovich, 2009; Henter et al., 2007).

There are two major subtypes of genetic causes of HLH. First are those genetic defects, grouped under the term FHL, that present with HLH as the primary and only manifestation of disease (Gupta & Weitzman, 2010; Henter et al., 2007). A second group of genetic disorders include HLH as only one, although often fatal, manifestation of the disease (Gupta & Weitzman, 2010; Janka, 2009). All known genetic abnormalities causing FHL involve genes that regulate proteins important in the secretory cytolytic pathway of NK-cells and CD8+ T lymphocytes. In 1999, the first FHL-inked locus was discovered on chromosome 9q21.3–22 in several Pakistani families and was later defined as the FHL1 subtype (Ohadi et al., 1999). Shortly thereafter, mutations in the perforin gene *PRF1* were discovered on chromosome 10q21 in a group of patients with FHL (FHL2 subtype) (Stepp et al., 1999). Furthermore, mutations in genes *UNC13D* (located on chromosome 17q25; FHL3 subtype)*, STX11* (located on chromosome 6q24; FHL4 subtype), and most recently *STXBP2* (located on chromosome 19p13; FHL5 subtype) were described (Feldmann 2003; zur Stadt et al., 2005, 2009). In view of the remarkable progress since the discovery of the first genetic defect in

Autoimmune-Associated Hemophagocytic Syndrome/Macrophage Activation Syndrome 95

intravenous immunoglobulin (IVIG), which may be sufficient to control hyperinflammation (Janka, 2009). In order to achieve rapid reversal of the coagulation abnormalities and cytopenias, most clinicians prefer starting with intraveneous methylprednisolone pulse therapy (30 mg/kg for 3 consecutive days) followed by 2 to 3 mg/kg/day divided by 4 doses (Filipovich et al., 2010). After improvement of the complete blood count and resolution of the coagulopathy, steroids are tapered slowly to avoid relapses of MAS (Janka, 2009; Filipovich et al., 2010). High-dose corticosteroids alone have been reported to induce remission in approximately half of MAS patients (Sawhney et al., 2001 Stephan et al., 2001). Administration of IVIG might be effective in AAHS/MAS. High dose IVIG infusions are immunosuppressive, in part engaging Fc-receptors, which can play an important role in same patients with autoimmune/autoinflammatory diseases (Arceci, 2008; Kumakura et al., 2004). IVIG may also provide an anti-pathogen effect, which is particularly important if

Even when treatment is introduced in a timely manner, MAS can be fatal and deaths have been reported among patients treated with massive doses of steroids (Filipovich et al., 2010). However, corticosteroid resistant non-responders may benefit from second-line therapies, such as CyA or etoposide. Parenteral administration of CyA has been shown to be effective in patients with corticosteroid-resistant MAS (Mouy et al., 1996; Ravelli et al., 1996). Of note, in author's experience, some patients with MAS have not responded until etoposide was added to the HLH therapy. The similar conclusion has recently been postulated by other authors (Gupta & Weitzman, 2010). Thus, if there is no response to the aforementioned drugs (corticosteroids, IVIG, CyA), use of the HLH-2004 protocol including etoposide is recommended (Table 9). In summary, patients with suspected AAHS/MAS could be started on therapy without etoposide, as long as treatment adjustments are made rapidly in

The utility of biological response modifiers in MAS treatment remains unclear, and at the present there is no consensus on recommendations in respect to this group of drugs. The use of TNF-α inhibitors (etanercept, infliximab) in MAS has produced conflicting results, being the effective therapy in some patients (Makay et al., 2008; Sellmer et al., 2011), while triggering MAS in others (Sandhu et al., 2007). Biological agents that neutralize IL-1 (anakinra) and IL-6 (tocilizumab) have been reported to be effective in occasional MAS patients (Filipovich et al., 2010; Kelly & Ramanan, 2008), but the clinical experience is as yet limited. In the case of patients with a form of sHLH other than AAHS/MAS, which proved refractory to frontline HLH therapy, anecdotal reports on the beneficial use of plasma exchange, hemofiltration, antithrombin III, anti-CD52 antibodies (alemtuzumab), and anti-CD25 antibodies (daclizumab) have been published previously, but the role of these therapies is not yet validated for any type of HLH (Gupta & Weitzman, 2010). Lastly, if MAS is driven by EBV infection, monoclonal anti-CD20 antibodies (rituximab) which deplete B lymphocytes, the main type of cells harboring EBV virus, should be used (Balamuth et al.,

The first successful allogeneic bone marrow transplantation in a case of HLH was reported in 1986 (Fischer et al., 1986). Since then, information regarding the role of alloSCT in the treatment of HLH has mostly concerned FHL (Janka, 2009; Marsh et al., 2010). In FHL, alloSCT is the only available curative treatment option with the reported 5-year overall

MAS is triggered by a viral infection.

refractory cases (Gupta & Weitzman, 2010).

2007).

**9.2 Stem cell transplantation** 

FHL in 1999, it is expected that many new mutations in the known genes will be identified, as well as some novel gene mutations (Gupta & Weitzman, 2010).

Viruses, non-steroidal anti-inflammatory drugs, methotrexate, gold salts, and even TNF-α inhibitors have been reported as triggers for AAHS/MAS (Gupta & Weitzman, 2010). Interestingly, distinctions between genetically determined and acquired HLH become increasingly blurred as brand new genetic causes are identified, and patients who develop HLH beyond early childhood or in the contexts of EBV infection or autoimmune disease are being found to share some of the same genetic etiologies (Arceci, 2008; Hazen et al., 2008; Nagafuji et al., 2007; Zhang et al., 2008). Patients who develop sHLH may also have a genetic predisposition, but the molecular basis of the defects in sHLH has yet to be discovered (Arceci, 2008). This supposition has recently been strengthened by recent studies showing decreased NK cell function or reduced perforin expression in children with sJIA complicated by MAS, similarly to patients with FHL (Grom et al., 2003; Wulffraat et al., 2003). Of note, mutations in *UNC13D* gene, mutated in FHL type 3, were also described in patients with sJIA (Hazen et al., 2008; Zhang et al., 2008).
