Familial hemophagocytic lymphohistiocytosis (FHL) was first described by Farquhar and Claireaux as familial erythrophagocytic lymphohistiocytosis 29. The incidence of FHL has been estimated to be 1 in 50,000 births 3031. Overwhelming HS is the distinguishing and isolated feature in this disorder; there are no other associated signs, unlike the other inherited forms. Symptoms of HS are usually evident within the first 3 months of life and can even develop in utero. Rare cases with delayed onset have been observed. HS most often occurs in previously healthy young children, which suggests the need for an exogenous trigger prior to the onset of clinical manifestations. In susceptible children, infection with intracellular pathogens (viral and fungal, among others) is the most likely trigger for disease manifestation 32. HS in FHL is invariably lethal unless treatment with allogeneic stem-cell transplantation is performed 33. Previously, linkage analysis using homozygosity mapping in four hereditary FHL families of Pakistani descent identified a locus (FHL1) on chromosome 9q21.3-22 34. However, no causative gene has been so far associated with this locus. Association of this locus with FHL seems restricted to Pakistani families although not all FHL cases in Pakistani families segregate with this locus 35. Using genome wide linkage analysis, two additional loci have been identified on chromosomes 10q21-22 (FHL2) 36 and 17q25 (FHL3) 35, and there is further evidence of additional genetic heterogeneity and of a yet-undefined gene or genes (G. de St Basile, unpublished data).

The cytolytic effector perforin, present in cytotoxic granules, was the first gene identified as causing FHL 37. As a consequence of perforin gene mutations, perforin protein expression is diminished to barely detectable in cytotoxic granules 233738, leading to defective cytotoxic activity. In normal cells, following release from lytic granules, perforin is thought to oligomerize in order to form a pore-like structure in the target cell membrane, analogous to the C9 component of complement 22. Failure of perforin activity is etiologically linked to the development of FHL, and its deficiency accounts for one-third of patients with FHL.

Patients whose disease is associated with FHL3 locus present typical features of FHL and are indistinguishable from patients with a perforin (ie, FHL2) defect. In patients with FHL3, however, perforin is normally expressed and is functional. FHL3 was found to be associated with mutations in the gene UnC13D encoding for hMunc13-4, a member of the Munc13-UNC13 family 35. Six different hMunc13-4 mutations have so far been identified in patients with FHL3 from seven different families. Studies of the exocytosis of cytotoxic granules in lymphocytes from patients with FHL3 mutations showed that Munc13-4 is required for the release of the lytic granule contents but not for other secretory pathways, including the secretion of IFN-γ from T cell antigen receptor (TCR)-activated lymphocytes 35. Thus, hMunc13-4 is an essential effector of the cytolytic granule pathway. Munc13-4-deficient lymphocytes can make normal contacts with target cells, stable conjugates, and polarize the lytic machinery as effectively as do control lymphocytes. However, when Munc 13-4 is lost in CTLs, cytotoxic granules dock at the membrane in the immunologic synapse but are not released (see Figure 2B). This supports a role for Munc 13-4 at a late step of this pathway in exocytosis subsequent to docking. Munc13-4 is most probably required at a priming step of lytic granule secretion, following granule docking and preceding plasma granule membrane fusion 243940. Of interest, Munc13-4 is expressed in numerous cell type, including platelets and lungs; however, the phenotype of patients with FHL3 is not different from that of patients with perforin deficiency.

Perforin and Munc 13-4 deficiencies account for only two-thirds of patients with FHL. Other genes are certainly involved and need further investigation. Analyses of new hereditary families with affected siblings that harbour no perforin or Munc13-4 mutations are needed and should outline other genes that are responsible for FHL.

Distinguishing primary forms from secondary forms of HS is important not only in terms of genetic counselling for this condition but also for determining the appropriate therapeutic intervention. The occurrence of HS at a young age should instigate the search for a genetic cause. Microscopic analysis of the hair shaft is an easy and reliable test for diagnosing Griscelli syndrome and Chédiak-Higashi syndrome. In both conditions, pigmentation dilution is characteristic, but there is larger clumping of pigment in the hair shafts of a patient with Griscelli syndrome than in the hair shafts of a patient with Chédiak-Higashi syndrome (see Figure 4B). Carriers of these syndromes have normal pigmentation. The presence of giant intracytoplasmic granules in all cells from the hematopoietic lineage is a hallmark of Chédiak-Higashi syndrome; this is easy to identify in a blood smear (see Figure 4C) and rapidly ensures diagnosis. If pigmentation dilution orients toward Griscelli syndrome, sequencing of the RAB27A gene allows confirmation of that diagnosis. In the absence of HS, molecular diagnosis of Griscelli syndrome is important for ruling out potential RAB27Adeficiencies, which should be treated by allogeneic stem cell transplantation. In Chédiak-Higashi syndrome, given the length of the CHS1 gene, mutation screening is not used as a routine test for diagnosis and genetic counselling. An unambiguous diagnosis of this condition can be made without need for further genetic testing, based on the characteristic hypopigmentation of hair shafts and the presence of intracellular giant granules. However, for genetic counselling of families, segregation analysis of polymorphic markers linked to the Chédiak-Higashi syndrome locus on chromosome 1q43.2 in the family can be used. In nonconsanguineous families, this approach requires the availability of a sample of deoxyribonucleic acid (DNA) from both parents and from the patient to determine the affected haplotype in the family. When parents are related, the identification of a shared haplotype at the Chédiak-Higashi syndrome locus in the parents may overcome the unavailability of a DNA sample from the patient. When HS is not associated with hypopigmentation, the biggest difficulty lies in differentiating between the primary (inherited) disease (FHL) and a secondary HS disease. A positive family history with previously affected family members and/or consanguinity of the parents is highly suggestive of an inherited form. The availability of biologic samples from family members such as parents and siblings greatly helps the molecular diagnosis of genetic causes by rapid determination of the polymorphic markers segregating with the disease locus. However, the lack of family history is not a reliable criterion for excluding FHL. The study of the cytotoxic activity of T lymphocytes 3740 is a reliable test with which to diagnose the genetic forms of HS. About 30% of FHL cases result from a perforin defect, which can be rapidly identified by immunofluorescence analysis of perforin expression in resting cytotoxic cells. In fact, the great majority of mutations so far identified in FHL2 dramatically affect perforin detection. Sequencing of the perforin gene will confirm the diagnosis of FHL. Another group of FHL cases (about 60%) is characterized by defective T-cell cytotoxic activity but normal perforin expression. In half of these cases, sequencing of the MUNC13.4 gene allows identification of FHL from Munc13-4 deficiency. In the rest, the genetic cause is not yet characterized. Defective T-lymphocyte cytotoxic activity is the signature of a primary genetic cause of HS in 90% of cases. In approximately 10% of FHL cases, however, defects in T-cell cytotoxic activity cannot be evidenced. In the absence of family history, these forms cannot be clearly distinguished from secondary forms of HS, and they remain a diagnostic challenge.

Finally, the diagnosis of X-linked lymphoproliferative syndrome should be confirmed by sequencing of the SAP gene and potentially by the analysis of SAP protein expression, with the knowledge that a significant number of patients with the X-linked lymphoproliferative syndrome-like phenotype do not have mutations in this gene but potentially do have mutations in other yet-uncharacterized genes 5960.

In the early 1980s, several reports described patients with systemic-onset juvenile rheumatoid arthritis (JRA) in whom a severe coagulopathy resembling disseminated intravascular coagulation developed 65. Such a coagulopathy was often associated with changes of mental status, hepatosplenomegaly, increased serum levels of liver enzymes, and sharp falls in blood counts and erythrocyte sedimentation rates. In 1985, Hadchouel and colleagues linked these symptoms to massive proliferation of activated nonneoplastic macrophagic histiocytes with prominent hemophagocytic activity 66. The term macrophage activation syndrome (MAS) was eventually introduced in 1993 by Stephan and colleagues in a follow-up report originating from the same centre 5. Over the following years, several more reports from various countries described a number of patients with very similar symptoms. MAS, reactive hemophagocytic lymphohistiocytosis (HLH), and HS are different denominations of the same clinical entity. Although HS has also been observed in a small number of patients with polyarticular JRA and in those with collagen diseases (including lupus, vasculitis, Kawasaki disease, dermatomyositis, and panniculitis), it is most commonly seen in patients with the systemic form of JRA 676869.

It is still unclear why some individuals with these rheumatologic disorders develop MAS during the course of their disease. A pathogen trigger is often present, initiating HS in this setting. In a study including seven patients with MAS, decreased NK-cell activity was observed in all patients, and decreased perforin expression was found in two of the seven patients despite a normal perforin 1 gene sequence 70. Decreased expression of SAP transcript has also been reported in peripheral T cells of patients with JRA 71. A transient inhibition of the cytotoxic granule pathway may be sufficient in a setting of immune system disturbances such as systemic JRA or other rheumatoid disorders. In these patients, pathogens may not be properly cleared during the first wave of infection, thus promoting the development of very potent immune system activation and HS. The hypothesis that impaired cytotoxic functions and lack of immunoregulation by NK cells result in MAS/HS in rheumatoid disorders remains to be proven. What is clear is that both inherited HS and acquired HS share the same immunologic abnormalities in terms of macrophage and T-lymphocyte activation and expansion and should initially be treated similarly, as overwhelming lymphocyte activation.

In 1979, HS was described in a cohort of patients who had serologic evidence of recent viral infections, and virus-associated HS was proposed as a distinct clinical entity 72. Subsequently, HS has been reported in association with a variety of infections, and the term "reactive hemophagocytic syndrome" has been suggested to distinguish HS associated with an identifiable infectious or noninfectious cause from its hereditary forms. However, the reactive and hereditary forms of the disease are difficult to distinguish; for example, patients with familial forms of HS may have HS after a documented infection. Case reports and case series on the association of infections and HLH have been summarized 26. The most frequent pathologic conditions that can cause HS are infection by viruses (including viruses of the herpes family, human immunodeficiency virus, and adenovirus), fungal infections, bacterial and mycobacterial infections, and parasitic infection (including visceral leishmaniasis and toxoplasmosis) 117374757677787980818283848586. Special emphasis should be given to EBV-associated HS. With the exclusion of inherited disorders, this form of acquired HS seems more severe than other types of infection-associated HS. It is a recurrent overwhelming HS, and its outcome is often fatal despite optimal management.

The etiology of this syndrome remains poorly understood (see below). The resolution of HS following treatment of the infection suggests that in many cases, HS is secondary to the underlying infection. A diagnosis that takes into account all of the underlying diseases associated with HS would be impractical, and formal guidelines for evaluating patients with suspected infection-associated HLH have not been established. Extensive testing for underlying infecting organisms should be guided by epidemiologic data and the patient's medical history.

HS has been shown to be associated with malignant histiocytosis 72. In regard to malignancies, HS is mostly encountered in patients with T-cell malignancies, especially T-cell lymphomas 878889. It can be present at onset or during the course of treatment and usually implies relapse or escape of the leukemic clone from chemotherapeutic agents 90. Although T lymphocytes lack the putative EBV receptor CD21, the presence of episomal EBV genome in T-cell lymphomas 25919293 and T lymphocytes from patients with virus-associated HS is has been well described 94. EBV-positive T-cell lymphomas appear to elaborate TNF-α more frequently than either EBV-positive B-cell lymphomas or EBV-negative T-cell lymphomas 2593. Lay and colleagues induced the expression of CD21 in T-lymphoma cell lines and subsequently infected these cells with EBV. High levels of TNF-α, IFN-γ, and IL-1α were secreted by these cells after EBV infection; when the lymphocytes were cocultured with monocytes, enhanced phagocytosis by monocytes was observed. The enhanced phagocytosis was eliminated by the addition of antibodies against TNF-α and IFN-γ 2593. Clonal expansion of EBV-infected T lymphocytes has been demonstrated in both EBV-associated HS and EBV-positive T-cell lymphoma by the presence of homogeneous viral terminal repetitive sequences. EBV-infected cells stain positive for such T-lymphocyte markers as CD45RO and TCR-β. Clonality of infected T lymphocytes is further suggested by the finding of monoclonal re-arrangements of the TCR-β gene in EBV-associated HS 95. The distinction between the monoclonal proliferation of T lymphocytes seen in EBV-associated HS and EBV-positive T-cell lymphomas may describe the extremes of a spectrum of disordered T-lymphocyte proliferation and cytokine elaboration following EBV infection of T lymphocytes. It is unclear whether clonal proliferation of T lymphocytes occurs in HS that is associated with pathogens other than EBV. The fact that these syndromes seem more likely to resolve with control of the underlying infection suggests that this may not be the case 72.

The treatment strategy for MAS/HS is based on the parenteral administration of high doses of methylprednisolone 67. After normalization of coagulation and hematologic abnormalities, a slow taper is then performed. Frequently, MAS is resistant to corticosteroids, and cases with fatal evolutions despite the administration of massive doses of steroids have been described 67. Parenteral administration of cyclosporin A at a dosage of 2 to 8 mg/kg/d has revolutionized outcomes 48799. There are anecdotal reports on the use of etoposide and intravenous immunoglobulins for treatment of MAS; the results conflict, and we do not suggest the use of these drugs in MAS cases. Therapies that inhibit T-cell function may be expected to make patients with JRA more susceptible to the development of HS in the face of infection; on theoretical grounds, therefore, one may recommend that such drugs be reduced transiently during episodes of infection.

Ashort course of intravenous methylprednisolone (1-2 mg/kg/d) is often sufficient in treating HS associated with bacterial, parasitic, and fungal infections. This treatment is given in concert with adequate antiinfectious agents, and it must be administered for the shortest time necessary for correction of the most life-threatening symptoms associated with HS because of the added immunosuppression in the context of severe infection. EBV-associated HS remains a therapeutic challenge and often has a fatal outcome despite aggressive management. We suggest that this form be treated as HS associated with inherited disorders. New antiviral drugs such as cidofovir or ribavirin may be useful; however, the results achieved with these drugs in the treatment of other B lymphoproliferative disorders associated with EBV are conflicting. Consideration should be given to the use of IVIG in infection-associated HS, to minimize the risk of immunosuppression.

In malignancies, a short pulse of intravenous steroids (2 mg/kg/d) is usually administered along with conventional chemotherapy adapted to the underlying disease. With the destruction of the malignant clone, resolution of HS is often seen and no additional treatment is needed. Etoposide (100 mg/m² per dose) as per the HLH-94 protocol 96 can be considered in case of resistance to corticosteroids.