Donna Horncastle

Mycobacterium tuberculosis Up-Regulates Matrix Metalloproteinase-1 Secretion from Human Airway Epithelial Cells via a p38 MAPK Switch

Pulmonary cavitation is vital to the persistence and spread of Mycobacterium tuberculosis (MTb), but mechanisms underlying this lung destruction are poorly understood. Fibrillar type I collagen provides the lung’s tensile strength, and only matrix metalloproteinases (MMPs) can degrade it at neutral pH. We investigated MTb-infected lung tissue and found that airway epithelial cells adjacent to tuberculosis (Tb) granulomas expressed a high level of MMP-1 (interstitial collagenase). Conditioned media from MTb-infected monocytes (CoMTb) up-regulated epithelial cell MMP-1 promoter activity, gene expression, and secretion, whereas direct MTb infection did not. CoMTb concurrently suppressed tissue inhibitor of metalloprotease-1 (TIMP-1) secretion, further promoting matrix degradation, and in Tb patients very low TIMP-1 expression was detected. MMP-1 up-regulation required synergy between TNF-α and G protein-coupled receptor signaling pathways. CoMTb stimulated p38 MAPK phosphorylation, and this is the point of TNF-α synergy with G protein-coupled receptor activation. Furthermore, p38 phosphorylation was the switch up-regulating MMP-1 activity and decreasing TIMP-1 secretion. Activated p38 localized to MMP-1-secreting airway epithelial cells in Tb patients. These data reveal a monocyte-epithelial cell network whereby MTb may drive tissue destruction, and they demonstrate that p38 phosphorylation is a key regulatory point in the generation of a matrix-degrading phenotype.

In vivo evidence for apoptosis in the bone marrow in systemic lupus erythematosus

An increase in leucocyte apoptosis and impaired clearance of apoptotic cells has been observed in patients with systemic lupus erythematosus (SLE). Apoptotic cells are likely to be a key source of autoantigens in SLE as they express many of the nuclear autoantigens (in surface blebs and apoptotic bodies) that are relevant to this disease. The clearance of apoptotic cells is usually a rapid process, such that few cells are usually seen in the extracellular environment in vivo. We report a case in which multiple apoptotic bodies were observed in the bone marrow of a patient with SLE that was complicated by an immune-mediated pancytopenia. We have subsequently examined the frequency of apoptotic cells, identified morphologically, and by caspase-3 staining in bone-marrow trephine samples taken from patients with SLE over a 10-year period of follow-up. A high proportion of bone marrows contained apoptotic debris. The novel demonstration of apoptotic bodies in vivo in patients with SLE is unusual and supports the notion that the marrow may be a target organ in the disease. Their abundance is also consistent with the hypothesis that normal clearance mechanisms are defective and/or overwhelmed in SLE.

IFNγ synergizes with IL-1β to up-regulate MMP-9 secretion in a cellular model of central nervous system tuberculosis

Matrix metalloproteinase‐9 (MMP‐9) activity is implicated in pathogenesis of central nervous system tuberculosis (CNS‐TB). IFNγ, a key cytokine in TB, usually inhibits MMP‐9 secretion. Addition of IFNγ to conditioned media from M. tb‐infected monocytes (CoMTB) resulted in a 7‐fold increase in MMP‐9 activity detected by gelatin zymography (P<0.01). In contrast, tissue inhibitor of metalloproteinase (TIMP)‐1 and ‐2 secretion, measured by ELISA, was suppressed. Dexamethasone abolished the synergistic increase in MMP‐9 activity. Interleukin (IL)‐1β in CoMTB is a critical mediator of synergy with IFNγ, and IL‐1β alone synergizes with IFNγ to increase MMP‐9 secretion from 51 ± 31 to 762 ± 136 U. IL‐1β activity is dependent on p38 mitogen‐activated protein (MAPK) kinase, which was found to be phosphorylated in tissue specimens from patients with CNS‐TB. Extracellular signal regulated kinase (Erk) and p38 MAPK activation did not affect IFNγ signaling pathways. Inhibition of janusactivated kinase (JAK)‐2 by 50 μM AG540 decreased MMP‐9 secretion to 124 ± 11.1 from 651 ± 229 U of activity (P<0.01). However, signal transducer and activator of transcription (STAT)‐3 but not STAT‐1 phosphorylation was synergistically up‐regulated by IFNγ and CoMTB. In summary, synergy between IL‐1β and STAT‐3 dependent IFNγ signaling is key in control of up‐regulation of MMP‐9 activity in CNS‐TB and may be a significant mechanism of brain tissue destruction.—Harris, J. E., Fernandez‐Vilaseca, M., Elkington, P. T. G., Horncastle, D. E., Graeber, M. B., and Friedland, J. S. IFNα synergizes with IL‐1β to upregulate MMP‐9 secretion in a cellular model of central nervous system tuberculosis. FASEB J. 21, 356–365 (2007)

Monocyte-Astrocyte Networks Regulate Matrix Metalloproteinase Gene Expression and Secretion in Central Nervous System Tuberculosis In Vitro and In Vivo

CNS tuberculosis (CNS-TB) is the most deadly form of tuberculous disease accounting for 10% of clinical cases. CNS-TB is characterized by extensive tissue destruction, in which matrix metalloproteinases (MMPs) may play a critical role. We investigated the hypothesis that Mycobacterium tuberculosis activates monocyte-astrocyte networks increasing the activity of key MMPs. We examined the expression of all human MMPs and the tissue inhibitors of metalloproteinases (TIMPs) in human astrocytes stimulated by conditioned medium from M. tuberculosis-infected monocytes (CoMTB). Real-time RT-PCR showed that gene expression of MMP-1, -2, -3, -7, and -9 was increased (p < 0.05). MMP-9 secretion was significantly up-regulated at 24 h and increased over 120 h (p < 0.01). MMP-1, -3, and -7 secretion was not detected. Secretion of MMP-2 was constitutive and unaffected by CoMTB. Astrocyte gene expression and secretion of TIMP-1 was not affected by CoMTB although TIMP-2 secretion increased 3-fold at 120 h. Immunohistochemical analysis of human brain biopsies confirmed that astrocyte MMP-9 secretion is a predominant feature in CNS-TB in vivo. Dexamethasone inhibited astrocyte MMP-9, but not TIMP-1/2 secretion in response to CoMTB. CoMTB stimulated the nuclear translocation of NF-κB, inducing a 6-fold increase in nuclear p65 and a 2-fold increase in nuclear p50. This was associated with degradation of IκBα and β within 30 min, persisting for 24 h. In summary, networks active between monocytes and astrocytes regulate MMP-9 activity in tuberculosis and astrocytes are a major source of MMP-9 in CNS-TB. Astrocytes may contribute to a matrix degrading environment within the CNS and subsequent morbidity and mortality.

Monocyte-Dependent Fibroblast CXCL8 Secretion Occurs in Tuberculosis and Limits Survival of Mycobacteria within Macrophages

CXCL8 is a chemokine that is implicated in the formation of tuberculous (TB) granulomas and in immunity to Mycobacterium tuberculosis (Mtb). Fibroblast chemokine secretion is important for modulating inflammatory responses in chronic lung disease and inflammatory arthritis but has not been investigated in the pathophysiology of TB. In this study, we used a cellular model to examine monocyte/macrophage-dependent stimulation of fibroblasts by Mtb in the regulation of chemokine secretion, particularly that of CXCL8. Human lung fibroblasts grown in collagen were stimulated with conditioned medium from Mtb-infected monocytes (CoMTb). CoMTb-induced prolonged dose-dependent, p38-mediated expression of stable CXCL8 mRNA by fibroblasts accompanied by a >10-fold increase in CXCL8 secretion (487 ± 88 ng/ml vs 48.6 ± 34 ng/ml in controls) at 120 h. Fibroblasts strongly expressed CXCL8 in vivo in human TB granulomas. Inhibition of TNF-α or IL-1 in CoMTb abrogated the induction of CXCL8 at a pretranscriptional level. CXCL8 secretion was NF-κB, C/EBP, and JNK dependent. Sustained NF-κB activation was demonstrated beyond 24 h in response to CoMTb. Exogenous CXCL8 reduced the survival of Mtb within macrophages, and inhibition of CXCL8 was associated with intracellular mycobacterial proliferation. These data show that fibroblasts have a previously unrecognized role in modulating inflammation in TB by their CXCL8-dependent contribution to cell recruitment and mycobacterial killing within the granuloma.

Absence of cyclin-D2 and Bcl-2 expression within the germinal centre type of diffuse large B-cell lymphoma identifies a very good prognostic subgroup of patients

Aims:  To validate and improve the existing algorithm (proposed by Hans et al.) to classify diffuse large B‐cell lymphoma (DLBCL).

Bone marrow trephine combined with immunohistochemistry is superior to bone marrow aspirate in follow-up of myeloma patients

Aims: Multiple myeloma (MM) guidelines in the UK do not advocate performing bone marrow trephine biopsy (BMTB) during follow-up. In a recent study, it was found that the plasma cell per cent (PC%) in BMTB performed at the time of autologous stem cell transplant strongly correlated with survival. The current study addresses whether BMTB is superior to bone marrow aspiration (BMA) in documenting presence of disease and its volume at follow-up. Methods: The study involved 106 samples. A conventional 500-cell differential count was performed on the BMAs to document the PC%. The PC% on BMTBs had been estimated on CD138 immunostain. Furthermore, BMTBs had also been immunostained for CD56, cyclin D1 and light chains. Results: The mean (2SEM) PC% values in BMAs and BMTBs were 13.1 (2.6)% and 31.8 (5.8)% respectively. Based on BMA, BMTB and serum/urine paraprotein or light chain estimation, on 92 occasions (89%) there was detectable disease. The positive predictive value of both BMA and BMTB was 100%, and the negative predictive values for BMTB and BMA were 57% and 22% respectively. Among 98 secretory MM cases, the BMTB-PC% showed significant correlation with paraprotein levels, whereas BMA-PC% did not. Conclusions: It is strongly recommended that BMTB is performed and adequately investigated with immunohistochemistry during follow-up of MM.

CD20 expression in the HHV‐8‐infected lymphoid cells in multicentric Castleman disease

peripheral T-cell lymphoma, unspecified, and anaplastic large T-cell lymphoma delineates novel recurrent chromosomal alterations. Am. J. Pathol. 2004; 164; 1837–1848. 2. Greer JP, Batt MA, Whitlock JA et al. Clinical features of the small cell variant (SCV) of Ki-1+ anaplastic large cell lymphoma (ALCL). Blood 1995; 86 (Suppl. 1); 532a. 3. Lesesve JF, Buisine J, Grégoire MJ et al. Leukaemic small cell variant anaplastic large cell lymphoma during pregnancy. Clin. Lab. Haematol. 2000; 22; 297–301. 4. Awaya N, Mori S, Takeuchi H et al. CD30 and the NPM-ALK fusion protein (p80) are differentially expressed between peripheral blood and bone marrow in primary small cell variant of anaplastic large cell lymphoma. Am. J. Hematol. 2002; 69; 200–204.

The expression of minichromosome maintenance protein-2 in normal and abnormal megakaryocytes and comparison with the proliferative marker Ki-67.

The minichromosome maintenance (Mcm) and Cdc6 proteins are important regulators of eucaryotic DNA replication. In most normal tissues, a similar proportion of cells express Mcm‐2 and Ki‐67. The present study showed that in both normal and abnormal states, the proportion of megakaryocytes expressing Mcm‐2 is roughly seven times as many as those that express Ki‐67. This is likely to be related to the process of endomitosis and endoreduplication. We also demonstrated that a significantly lower proportion of megakaryocytes in myelodysplastic syndrome express Mcm‐2. These findings provide new insights into megakaryocyte biology.

Bcl-6 and c-Myc are rarely co-expressed in adult diffuse large B-cell lymphoma.

Bcl-6 is expressed in germinal centre derived B-cell non-Hodgkin lymphomas including diffuse large B-cell lymphoma (DLBCL) and is likely to play a major role in driving proliferation of a subset of DLBCLs, especially those of germinal centre B-cell subtype, but the role of c-Myc, which is important for proliferation in various lineages is not known. We used the highly standardised staining conditions of a tissue microarray to characterise co-expression of c-Myc and Bcl-6 in DLBCL. We carried out immunohistochemistry of 73 arrayed cases. The majority (62/73) did not express c-Myc, but 11 cases (15%) showed nuclear staining. 5/53 (9%) of Bcl-6 expressing cases co-expressed c-Myc, whereas a much higher proportion, 6/20 (30%), of Bcl-6 negative cases were positive for c-Myc. Overall survival of c-Myc expressing cases was the same as those that had absent expression. There was no significant correlation between c-Myc expression and DLBCL subtype (germinal centre or non-germinal centre subtypes).