Familial disseminated atypical mycobacterial infection in childhood: a human mycobacterial susceptibility gene?

Familial disseminated atypical mycobacterial infection in childhood: a human mycobacterial susceptibility gene?

Summary

Introduction

Inherited defects in specific components of the immune system have provided many clues to the immunological mechanisms underlying resistance to microbial infection. We report a familial immune defect predisposing to disseminated atypical mycobacterial infection in childhood. 6 children with disseminated atypical mycobacterial infection and no recognised form of immunodeficency were identified. Four, including two brothers, come from a village in Malta, and two are brothers of Greek Cypriot origin. They presented with fever, weight loss, lymphadenopathy, and hepatosplenomegaly. They had anaemia and an acute phase response. A range of different mycobacteria (Mycobacterium fortuitum, M chelonei, and four strains of M avium intracellulare complex) were isolated. Treatment with multiple antibiotics failed to eradicate the infection, although treatment with gamma interferon was associated with improvement. Three have died and the surviving children have chronic infection. Tumour necrosis factor-&agr; production in response to endotoxin and gamma-interferon was found to be defective in affected patients and their parents. T-cell proliferative responses to mycobacterial and recall antigens were reduced in parents of affected children and gamma-interferon production was diminished in the affected patients and their parents. Clinical and immunological features suggest that these patients are phenotypically similar to Lsh/Ity/Bcg susceptible mice. Understanding of this defect may provide insights into the mechanisms responsible for susceptibility

One of the main unanswered questions in our understanding of mycobacterial infections is why disease occurs in only a small proportion (approximately 10%) of individuals who are infected. Atypical mycobacteria are ubiquitous and most people have positive skin tests or serological evidence of previous infection.1-3 With the exception of localised submandibular lymph node infection in childrenprogressive disease due to atypical mycobacteria is extremely rare in normal individuals. Immunological factors determine whether mycobacterial infection results in progressive clinical disease or in the development of an immune response that contains the

to

mycobacteria. Lancet 1995; 345: 79-83

infection.

Mycobacterial disease is most common at the extremes of age when immune responses to many pathogens are reduced4 and is increased malnutrition, by immunosuppressive drugs, or HIV infection. 5,6 Cellular and molecular mechanisms which distinguish a protective immune response from an ineffective response and progressive disease are poorly understood. In mice, resistance to Mycobacterium bovis is controlled by a dominant autosomal gene, Bcg.7 The same locus controls resistance to M leprae murium,8,9 M avium," and M intracellulare." The gene is believed to be identical to the Lsh and fty loci that control resistance to intracellular pathogens such as leishmania12 and salmonella."’"* The LshlftylBcg gene is expressed in the macrophage and is involved in regulation of macrophage priming and activation. 15,16 Macrophages from susceptible mice are defective in the production of toxic oxygen and nitrogen radicals in response to stimuli such as interferon and BCG.16-18 They also have diminished antigen-presenting capabilities,19 are not upregulated normally by gammainterferon, and the production of cytokines, including tumour necrosis factor-a (TNF-a), in response to

Departments of Paediatrics (Prof M Levin FRCP, M J Newport MRCP, Panos Kalabalikis MD, N Klein MRCP), Immunology (S D’Souza PhD), and Medical Microbiology (I N Brown PhD), St Mary’s Hospital Medical School, Imperial College of Science, Technology and Medicine, London W2 1PG, UK; Department of Paediatrics, St Luke’s Hospital, Guardamangia, Malta (H M Lenicker MD, P Vassallo Agius FRCP); Department of Child Health, St George’s Hospital, London (E G Davies FRCP); Department of Medicine, University College Hospital, London (A Thrasher MRCP); and University of Cambridge Clinical School, Department of Medicine, Addenbrooke’s Hospital, Cambridge, UK (Prof J M Blackwell PhD) Correspondence to: Professor Michael Levin

endotoxin and gamma-interferon is diminished.’" The LshlftylBcg gene has been localised to the proximal part of mouse chromosome 120,21 in a region that shows conserved synteny with a region on the long arm of human chromosome 222 leading to speculation that a human homologue might exist.23 A candidate gene has been identified in mice and named Nramp (naturalresistance-associated macrophage protein gene).24 Hydropathy analysis suggests Nramp encodes an integral membrane protein that has structural homology with known prokaryotic and eukaryotic transport systems,

suggesting

a

macrophage-specific membrane-transport

function. A human homologue of Nramp is now known exist,24 but its precise function remains unknown and

to to

79

date, no defect equivalent to that reported in L/7/J3csusceptible mice has yet been identified in humans. Between 1981 and 1989, four Maltese children suffered from

syndrome of disseminated atypical mycobacterial infection, not associated with a previously known immunodeficiency. As all four came from one village in Malta, and three were related, we speculated that they might be suffering from an inherited immune defect predisposing to mycobacterial infections. Two more children were subsequently identified with the same syndrome. an

unusual

Patients and methods Case histories 3 of the affected children are related (figure 1). Case 4 is not known to be related to the other three children. Cases 5 and 6 are brothers of Greek Cypriot origin.

Case 1 The male first child of healthy Maltese parents was well until the age of 1 year, when he had anorexia, listlessness, fevers, night sweats, and splenomegaly. He was anaemic with hypoalbuminaemia and raised inflammatory markers. Repeated cultures for bacterial infections were negative, and bone-marrow aspirate showed a reactive picture. Mantoux test was negative and rheumatoid factor positive. Symptoms showed no response to aspirin, antibiotics, or prednisolone. Biopsy of submandibular lymph nodes and liver showed a non-specific inflammatory infiltrate with no evidence of granuloma formation. Acid-fast organisms subsequently identified as M chelonei were found in the lymph nodes. He was treated with erythromycin and prednisolone but his condition deteriorated and progressive pneumonia resulted in his death at the age of 32. Case 2 The male first child of healthy Maltese parents, both of whom are related to the parents of case 1 (figure 1), had fever, night sweats, lymphadenopathy, and splenomegaly at the age of 3 years. Heaf test was positive; lymph node biopsy showed reactive hyperplasia and no acid-fast organisms. He developed erosion of the head of the left femur, collapse of his seventh thoracic vertebra, and sclerosis of several other vertebrae. A trial of antituberculous treatment and subsequently of aspirin did not improve his condition. Bone marrow aspiration and laparotomy were undertaken with biopsies of his liver and mesenteric lymph nodes. The histology showed non-specific inflammatory changes without granuloma formation. Stains for mycobacteria, fungi, and leishmania were negative. Further investigation showed an intense acute phase response and markedly increased IgG and IgM. Mantoux test was positive (20 mm). Technetium bone scan showed multiple areas of increased isotope uptake. Biopsy of the collapsed thoracic vertebra and hilar nodes showed non-specific inflammatory changes with no acid-fast organisms nor granulomas. He was again treated with anti-tuberculous drugs but his symptoms worsened. In view of the possibility of a non-infectious inflammatory condition, he was commenced on treatment with prednisolone. He improved until the dose was decreased. Cyclophosphamide was added, but 2 weeks later there was increasing hilar lymphadenopathy and a draining sinus developed over his sixth rib. Acid-fast organisms were present in material from this site and Mfortuitum was subsequently identified. He was treated with multiple anti-atypical mycobacterial

drugs, including amikacin, cefoxitin, clarithromycin, rifabutin, but his condition deteriorated. Gamma added to his treatment and his condition improved markedly. One year later, gamma-interferon was stopped, and despite continuation of anti-mycobacterial drugs, his condition with worsened progressive hepatosplenomegaly, again lymphadenopathy, wasting, and draining sinuses. M fortuitum, was persistently isolated from biopsies of lymphnodes, bone marrow, and sinuses. Re-introduction of gamma-interferon and trials of most available anti-microbial agents, has not prevented his progressive deterioration. and

clofazimine,

interferon

80

Figure 1: Partial pedigree showing affected Maltese children Affected individuals (cases 1 to 4)

are

represented by closed symbols.

Case 3 The brother of case 1 was well until the age of 15 months when he had fever, weight loss, lymphadenopathy, and hepatosplenomegaly. He was anaemic and had raised inflammatory markers. Diagnostic laparotomy was undertaken with biopsy of liver and mesenteric nodes. Ziehl-Nielsen staining of both tissues revealed small numbers of acid-fast bacilli without associated granulomas. M avium intracellulare was isolated from both specimens. He improved after treatment with combinations of anti-atypical mycobacterial drugs and gamma-interferon, which was administered for one year. Over the last two years his general condition has remained good, and he has grown and developed normally. However he has again developed increasing lymphadenopathy together with evidence of an acute phase response. Gamma interferon has been re-instituted and he continues to take 5 anti-atypical mycobacterials. Case 4 The female first child of unrelated Maltese parents and weight loss at the age of 2 years and 9 months. Ultrasound and computed tomography scan of her abdomen showed enlarged mesenteric and para-aortic lymphnodes. Laparotomy and biopsy of mesenteric lymph nodes showed histiocytic proliferation and marked neutrophil infiltration. Stains for acid-fast organisms and leishmania were negative. She was thought to have viral-driven histiocytic proliferation and was treated with prednisolone. There was an initial improvement with resolution of fever, but progressive enlargement of liver, spleen, and lymphnodes occurred. Repeat laparotomy showed massive mesenteric and retroperitoneal lymphadenopathy, biopsy of which showed a mixed inflammatory infiltrate and large numbers of acid-fast organisms within histiocytes. There was no granuloma or giant-cell formation. M avium intracellulare was isolated from mesenteric lymphnodes and peripheral blood. Despite treatment with multiple antimicrobial agents she remained febrile, wasted, and with transfusion dependent anaemia. She was commenced on gamma interferon, and thereafter improved, but went on to develop bilateral pulmonary infiltrates, progressive splenomegaly and hypersplenism requiring splenectomy. Histology of the spleen showed multiple non-caseating granulomata with giant-cell formation. She remained well for 2 years while continuing gamma-interferon and multiple antibiotics. At the age of 6 years she had salmonella septicaemia which was successfully treated. However she developed pneumococcal meningitis following which acid-fast bacilli were isolated from her cerebrospinal fluid. Despite treatment she died of progressive central nervous system infection.

developed fever, anorexia, diarrhoea,

was

Case 5 The first child of healthy, unrelated parents of Greek Cypriot origin developed cervical lymphadenopathy at the age of 6 Investigations showed neutrophil leucocytosis, years. thrombocytosis, and a raised erythrocyte sedimentation rate. Chest radiograph showed consolidation of the left upper lobe. Mantoux test was negative. Biopsy of the cervical lymphnodes showed non-caseating epithelioid granulomas with giant cells and numerous histiocytes. Acid-fast organisms were seen and

Figure 2: Ratios of TNF-a produced in response to stimulation gamma-interferon and endotoxin to TNF-a produced in

with

response to endotoxin alone Bars denote

mean

values for each group.

M avium intracellulare subsequently cultured from lymphnodes, sputum, and gastric washings. Despite treatment with several

anti-mycobacterial drugs, he developed splenomegaly, bronchial obstruction, weight loss, and opacification of both lung fields; he died 2 years later. Case 6 The brother of case 5 was well until the age of 11 when he had persistent cervical lymphadenopathy. Biopsy of the enlarged nodes showed a non-specific chronic inflammatory response without granuloma formation and salmonella species were isolated on culture of the node and his stool. Treatment with co-trimoxazole led to some improvement but not to complete resolution of the lymphadenopathy. He remained generally well but had a persistently raised erythrocyte sedimentation rate for the next 8 years. Salmonella enteritidis was still present when repeat lymph node biopsy was undertaken at the age of 18. At the age of 23 he developed progressively enlarging lymphnodes from which M avium was isolated. His general condition improved on anti-microbial treatment and gamma-interferon, although he has persistent lymphadenopathy.

monocyte TNF-a production); children with uncomplicated acute infections (used as controls in T-cell responses); healthy adults; and parents of the children used as controls for monocyte TNF-a production and as controls in T-cell responses. Venous blood was collected in one tenth volume of 3-8% trisodium citrate. Gamma-interferon (Boehringer Ingelheim, Bracknell, Berks, UK; final concentration 2 (JLg/mL) or an equivalent volume of phosphate buffered saline (PBS) was added to 0-5 mL aliquots of the whole blood in sterile endotoxin-free polypropylene tubes. The tubes were incubated at 37°C with constant rotation at 30 revolutions per min. After 2 h of incubation, Escherichia coli endotoxin in 10 ILL PBS (Sigma Chemicals, St Louis, MO, USA; final concentration of 1 ILg/mL) was added and incubation continued as before for 3 h more. Samples were then centrifuged (1200 g for 10 min) and plasma was recovered. TNF-a concentration in the plasma was then assayed with an enzyme-linked immunoassay as described.25 TNF-a produced in response to endotoxin plus gammainterferon was compared with that produced in response to endotoxin alone, gamma-interferon alone, or identically handled samples incubated only with equivalent volumes of PBS. Results were expressed as a ratio of TNF-a produced in response to stimulation with gamma-interferon and endotoxin to TNF-a produced in response to endotoxin alone.

T-cell

proliferative production

responses and gamma-interferon

Peripheral blood mononuclear cells (PBMCs) were recovered by density gradient centrifugation in Ficoll Hypaque (Pharmacia, Uppsala, Sweden). PBMCs were washed in RPMI 1640 (GIBCO, Grand Islands, NY, USA) and plated at a density of 1-2 X 105 cells per well in 200 jjbL RPMI 1640 culture medium supplemented with 5% A+serum, 2 mmol L-glutamine, 100 U/mL penicillin, and 100 jjbg/mL streptomycin. Cells were stimulated with PHA (1 ug/ml, Sigma Chemicals) for 3 days; or mycobacterial antigens prepared by sonication of the patient’s own mycobacterial isolate (10" 10’, or 105’mL), M tuberculosis soluble extract (20 mg/mL), or diptheria, tetanus, and pertussis antigens (DPT, 1:4000 and 1:8000 dilutions) for 7 days. Microcultures were pulse-labelled for 12-18 h with 0-037 MBq tritated thymidine (Amersham, Buckinghamshire, UK) before harvesting. Before pulsing the cells with tritiated thymidine, 20 )JbL of the supernatant was removed for gamma-interferon assay with a commercially available kit (Dynatech, Chantilly, VA, USA).

Immunological studies

Results

Total T cell numbers and T cell subsets were normal in all children as were B cells, natural killer cells, and monocytes. Responses to phytohaemagglutinin (PHA) were normal except during the initial investigations of cases 1 and 3 who had moderately reduced PHA responses while they were severely ill. PHA response in case 3 after treatment of the infection was normal. Nitroblue tetrazolium tests, neutrophil opsonisation, super-oxide generation, and neutrophil chemotaxis were normal. Neutrophil killing of candida and staphylococci was normal. IgG, IgA, and IgM were normal or increased. Immunoglobulin subclasses were normal. Specific antibody responses against common antigens were detected, and the levels of C3, C4, and total haemolytic complement were normal. Antibodies to HIV-1 and HIV-2 have been repeatedly negative.

The ratio of TNF-a produced in response to stimulation with gamma-interferon and endotoxin to TNF-a produced in response to endotoxin alone (mean [SD]) was lower in infected children (2-3 [0-6]) than in healthy adults (5-4 [2’34]) or children with localised atypical mycobacterial infection (5-63 [1-37]). The otherwise healthy parents of children with disseminated atypical mycobacterial infection also showed reduced TNF-a production (3-63 [1-63]) in response to gamma-interferon and endotoxin when compared with healthy adults or parents of control children (figure 2). Mean maximal T-cell proliferation in response to mycobacterial antigens in patients with disseminated atypical mycobacterial infection (mean 17098 counts per min) was similar to that in control children (mean 11281) (figure 3). T-cell proliferation to DTP antigens were similar in both affected (mean 13200) and control children (17541 counts per min). However, parents of children with disseminated atypical mycobacterial infection showed decreased T-cell proliferation to mycobacterial antigen (mean 9450) and DTP (6858 counts per min) when compared with healthy adults (23569 for mycobacterial antigens and 38500 counts per

Macrophage function To establish whether a defect in macrophage function, similar to that present in the Lsh/Ity/Bcg-susceptible mice occurred in these patients, a modification of the methods of Blackwell et a1’6 was used to investigate cytokine responses to endotoxin and gammainterferon. Six groups of patients and controls were studied: children affected by disseminated atypical mycobacterial infection; healthy parents of these children; otherwise-healthy children with uncomplicated submandibular lymph node infections due to atypical mycobacteria (used as controls for

81

defect in

Figure 3: Tritiated thymidine uptake (a) and gamma-interferon production (b) by peripheral blood mononuclear cells Bars represent

mean

value for each group.

min for DTP antigens) (figure 3). Gamma-interferon produced in response to mycobacterial antigens was reduced in both the affected children (mean 65-9 pg/mL) and their parents (80-34 pg/mL) compared with children with other infections (225 pg/mL) or healthy adults (460 pg/mL). Reduced gamma-interferon production was also observed in response to recall antigens in affected children and their parents (affected children 20-0 pg/mL, control children 311 pg/mL, Maltese parents 123 pg/mL, healthy adults 504 pg/mL).

Discussion These children appear to have a specific immunological defect pre-disposing to the development of dissseminated atypical mycobacterial infections and salmonella infections. Affected children presented with fever, wasting, lymphadenopathy, and hepatosplenomegaly. The illness with was associated anaemia, neutrophil leucocytosis, thrombocytosis; and elevation of erythrocyte sedimentation rate, C reactive protein and immunoglobulins. Several different mycobacterial species have been isolated, including Mfortuitum, M chelonei, and four different strains of M avium intracellulare. The infections have been difficult to treat, and despite administration of multiple combinations of anti-mycobacterial agents, three of the children died, and the others have persistent infection. The addition of gamma-interferon was associated with clinical improvement in 4 out of the 6 patients; granuloma formation (absent previously) has been observed after gamma-interferon treatment in the 2 children. the to establish underlying Investigations have not shown a defect recognised defect immunological in T-cell or B-cell function, immunoglobulin production, neutrophil function, or non-specific humoral immunity. Because the spectrum of pathogens affecting these patients was similar to that in mice homozygous for the LshlltylBcg susceptible allele, we looked for a similar 82

function with similar experimental We have shown that TNF-a production in protocols. to endotoxin and response gamma-interferon is reduced in both the affected patients and their parents as compared with controls. Furthermore, gamma interferon production in response to mycobacterial antigens, or the common recall antigens in DTP, were also diminished in both affected patients and their parents, despite normal PHA responses, T-cell proliferative responses to mycobacterial antigens or recall antigens were reduced in the parents of infected patients although the patients themselves overlapped the control range. As the patients are chronically infected, and were receiving gammainterferon at the time of study, their T cells may have been maximally stimulated already. Reduced responses in otherwise healthy Maltese parents are particularly informative as they, like the adult controls, are not being treated with gamma-interferon and are not chronically infected. Diminished T-cell proliferation might be due to either a defect in antigen presentation or processing, or in T cell function. However the diminshed TNF-a responses in response to endotoxin and the failure of upregulation of these responses by gamma interferon are reminiscent of the pleiotropic effects of the mouse Lsh/lty/Bcg gene, suggesting that the defect resides within the macrophage. In order to test the hypothesis that the defect in the Maltese children is due to a mutation in the human homologue of the Lsh/lty/Bcg gene we studied a number of polymorphisms in the region of interest on human chromosome 2. We have been unable to demonstrate linkage between the human locus, and the defect in these children (manuscript in preparation). Although a candidate gene, designated Nramp, responsible for the Lsh/lty/Bcg defect has now been identified24, its function is unknown. By analogy to other defects in cellular activation or signal transduction such as chronic granulomatous disease 26, it is possible that the protein encoded by Nramp is only one of a series of proteins required for normal macrophage activation. A phenotype similar to that of the Lsh/Ity/Bcg-susceptible mouse may therefore arise from defects in more than one gene. Support for this hypothesis comes from the observation that transgenic mice lacking the 55 kilodalton TNF receptor show phenotypic similarities to LshlltylBcgsusceptible mice in having diminished TNF responses to endotoxin, and defective killing of intracellular pathogens.27 Our patients may therefore have a defect phenotypically similar to that of Lsh/Ity/Bcg-susceptible mice, but due to mutation in alternative genes. Most immunological defects result in infections with a wide range of different pathogens. A remarkable feature of the patients reported here is that, despite a prolonged period of follow-up, including periods when they have been severely wasted and undergoing prolonged treatment through indwelling intravascular cannulae, they have not suffered from recurrent bacterial infections other than those due to mycobacteria or salmonella, with the exception of case 4 who had an episode of pneumococcal meningitis (possibly related to her previous splenectomy). Normal responses to common childhood infections over many years of observation suggest that the defect in these children is specific for mycobacteria and salmonella and that responses to other bacterial and viral pathogens are normal. There

macrophage

are

mycobacterial

reports infection

of

disseminated atypical occurring in more than one

member of a family. 21>-30 Uchiyama et al reported two M with avium intracellulare complex infection in siblings whom a monocyte defect was shown but not clearly defined.29 Holland et al reported a heterogeneous group of patients with disseminated non-tuberculous mycobacterial infection which included three male members of one family in whom gamma-interferon production by mononuclear cells was defective .28 Infection in this family was associated with other underlying conditions and the average age of onset was higher than in the patients we have described, suggesting the underlying mechanisms are different. Although the parents of the affected patients are themselves healthy, they have been shown to have defective TNF and gamma-interferon production similar to that occurring in affected children. Although the parents do not appear to have increased susceptibility to atypical mycobacteria, it is possible that they may be more susceptible to virulent mycobacteria such as M tuberculosis. An intriguing question is whether are an heterozygotes apparently healthy population who may develop progressive disease due to salmonella,

leishmania,

or

mycobacteria.

Identification of the cellular and molecular mechanisms underlying the defect in these children may provide clues to the wider problem of susceptibility to mycobacteria and other intracellular pathogens. M Newport is supported by the St Mary’s Hospital Medical School Special Trustees. Dr D’Souza was supported by a Wellcome Trust training

fellowship. References 1

2

3

4 5

6 7

8

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