Immunoglobulin free light chains are biomarkers of poor prognosis in basal-like breast cancer and are potential targets in tumor-associated inflammation.

Inflammation is an important component of various cancers and its inflammatory cells and mediators have been shown to have prognostic potential. Tumor-infiltrating mast cells can promote tumor growth and angiogenesis, but the mechanism of mast cell activation is unclear. In earlier studies, we demonstrated that immunoglobulin free light chains (FLC) can trigger mast cells in an antigen-specific manner. Increased expression of FLC was observed within stroma of various human cancers including those of breast, colon, lung, pancreas, kidney and skin, and FLC expression co-localized with areas of mast cell infiltration. In a large cohort of breast cancer patients, FLC expression was shown associated with basal-like cancers with an aggressive phenotype. Moreover, lambda FLC was found expressed in areas of inflammatory infiltration and its expression was significantly associated with poor clinical outcome. Functional importance of FLCs was shown in a murine B16F10 melanoma model, where inhibition of FLC-mediated mast cell activation strongly reduced tumor growth. Collectively, this study identifies FLCs as a ligand in the pro-tumorigenic activation of mast cells. Blocking this pathway may open new avenues for the inhibition of tumor growth, while immunohistochemical staining of FLC may be helpful in the diagnosis and prognosis of cancer.

Immunoglobulinfree light chains reduce in an antigen-specific manner the rate of rise of action potentials of mouse non-nociceptive dorsal root ganglion neurons.

Plasma B cells secrete immunoglobulinfree light chains (IgLC) which by binding to mast cells can mediate hypersensitivity responses and are involved in several immunological disorders. To investigate the effects of antigen-specific IgLC activation, intracellular recordings were made from cultured murine dorsal root ganglion (DRG) neurons, which can specifically bind IgLC. The neurons were sensitized with IgLC for 90min and subsequently activated by application of the corresponding antigen (DNP-HSA). Antigen application induced a decrease in the rate of rise of the action potentials of non-nociceptive neurons (MANOVA, p=2.10(-6)), without affecting the resting membrane potential or firing threshold. The action potentials of the nociceptive neurons (p=0.57) and the electrical excitability of both types of neurons (p>0.35) were not affected. We conclude that IgLC can mediate antigen-specific responses by reducing the rate of rise of action potentials in non-nociceptive murine DRG neurons. We suggest that antigen-specific activation of IgLC-sensitized non-nociceptive DRG neurons may contribute to immunological hypersensitivity responses and neuroinflammation.

Antigen binding characteristics of immunoglobulin free light chains: crosslinking by antigen is essential to induce allergic inflammation.

Beside the production of complete immunoglobulins IgG, IgE, IgA, IgM and IgD, consisting of tetrameric heterodimers of immunoglobulin heavy and light chains, B cells also secrete immunoglobulin free light chains (Ig-fLC). Previous studies showed that Ig-fLCs are able to induce immediate hypersensitivity reactions. It is apparent that recognition and binding of antigen are crucial steps in the onset of these inflammatory responses. In this study, the binding characteristics of Ig-fLC to antigen were further investigated using various biochemical approaches. In addition, we investigated whether antigen-mediated crosslinking of Ig-fLC is required to initiate allergic skin inflammation in vivo. Our study shows that binding of Ig-fLCs to antigen can be measured with different experimental setups. Surface plasmon resonance analysis showed real-time antigen binding characteristics. Specific antigen binding by Ig-fLCs was further detected using immunoblotting and ELISA. Using the ELISA-based assay, a binding affinity of 76.9±3.8 nM was determined for TNP-specific Ig-fLC. Antigen-induced ear swelling in mice passively sensitized with trinitrophenol-specific Ig-fLC was inhibited when multivalent antigen was combined with excess of monovalent antigen during challenge. We conclude that Ig-fLCs are able to interact with antigen, a prerequisite for antigen-specific cellular activation. In analogy to antigen-specific Fc receptor-induced mast cell activation, crosslinking of Ig-fLCs is necessary to initiate a local allergic response.

An association between neutrophils and immunoglobulin free light chains in the pathogenesis of chronic obstructive pulmonary disease.

RATIONALE: Neutrophils are key players in chronic obstructive pulmonary disease (COPD), and increased numbers of neutrophils are present in sputum and lung tissue of patients with COPD. Interestingly, immunoglobulin free light chains (IgLC) are able to prolong the life of neutrophils; therefore, IgLC may contribute to the chronic state of inflammation. OBJECTIVES: In this study, the relation between IgLC and COPD has been investigated. METHODS: We investigated the presence of IgLC in different murine lung emphysema models. IgLC levels in serum from mice and patients with COPD were examined by Western blot analysis and ELISA, respectively. IgLC levels in lung tissue were determined by immunohistochemistry. Fluorescence-activated cell sorter and immunofluorescent analysis were used to detect binding between IgLC and human neutrophils. Interleukin-8 (CXCL8) release by neutrophils after IgLC incubation was measured by ELISA. The effect of F991, an IgLC antagonist, was examined on the neutrophil influx in murine lungs after 5 days of smoke exposure. MEASUREMENTS AND MAIN RESULTS: Increased levels of IgLC in serum of cigarette smoke-exposed and cigarette smoke extract-treated mice compared with control mice were observed. Patients with COPD showed increased serum IgLC and expression of IgLC in lung tissue compared with healthy volunteers. Interestingly, IgLC bound to neutrophils and activated neutrophils to release CXCL8. F991 inhibited the IgLC binding to neutrophils and reduced the smoke-induced neutrophil influx in murine lungs after smoke exposure. CONCLUSIONS: This study describes for the first time an association between neutrophils and IgLC in the pathophysiology of COPD, which could open new avenues to targeted treatment of this chronic disease.

Cardiac Renin levels are not influenced by the amount of resident mast cells.

To investigate whether mast cells release renin in the heart, we studied renin and prorenin synthesis by such cells, using the human mast cell lines human mastocytoma 1 and LAD2, as well as fresh mast cells from mastocytosis patients. We also quantified the contribution of mast cells to cardiac renin levels in control and infarcted rat hearts. Human mastocytoma 1 cells contained and released angiotensin I-generating activity, and the inhibition of this activity by the renin inhibitor aliskiren was comparable to that of recombinant human renin. Prorenin activation with trypsin increased angiotensin I-generating activity in the medium only, suggesting release but not storage of prorenin. The adenylyl cyclase activator forskolin, the cAMP analogue 8-db-cAMP, and the degranulator compound 48/80 increased renin release without affecting prorenin. Angiotensin II blocked the forskolin-induced renin release. Angiotensin I-generating activity was undetectable in LAD2 cells and fresh mast cells. Nonperfused rat hearts contained angiotensin I-generating activity, and aliskiren blocked approximately 70% of this activity. A 30-minute buffer perfusion washed away >70% of the aliskiren-inhibitable angiotensin I-generating activity. Prolonged buffer perfusion or compound 48/80 did not decrease cardiac angiotensin I-generating activity further or induce angiotensin I-generating activity release in the perfusion buffer. Results in infarcted hearts were identical, despite the increased mast cell number in such hearts. In conclusion, human mastocytoma 1 cells release renin and prorenin, and the regulation of this release resembles that of renal renin. However, this is not a uniform property of all mast cells. Mast cells appear an unlikely source of renin in the heart, both under normal and pathophysiological conditions.

Free immunoglobulin light chains: a novel target in the therapy of inflammatory diseases.

In recent years, novel therapeutic strategies have become available for the treatment of chronic inflammatory disease. Neutralizing proinflammatory mediators such as leukotrienes and TNF-alpha, in addition to anti-IgE therapies (Omaluzimab) that target higher in the inflammatory cascade, have shown success in the treatment of allergic or autoimmune disorders. Free immunoglobulin light chains, which are produced by B lymphocytes and secreted into serum, might play a crucial role in the pathogenesis of inflammatory disease. Concentrations of free light chains are significantly increased under diverse pathological conditions in humans, and have been linked to the progression and severity of immune diseases. Here we discuss the importance of free immunoglobulin light chains as a potential therapeutic target in the treatment of chronic inflammatory disease.

Expression of tumor-associated differentiation antigens, MUC1 glycoforms and CEA, in human thymic epithelial cells: implications for self-tolerance and tumor therapy.

Expression of tissue-restricted self-antigens in the thymus, termed promiscuous gene expression, imposes T cell tolerance and protects from autoimmune diseases. This antigen pool also includes various types of tumor-associated antigens (TAA) previously thought to be secluded from the immune system. The scope of promiscuous gene expression has been defined by mRNA analysis at the global level of isolated medullary thymic epithelial cells (mTECs). Information at the protein level on the frequency of mTECs expressing a given antigen, on coexpression patterns, and post-translational modifications is largely missing. We report here promiscuous expression at the protein level of two TAA, MUC1 and CEA, in situ and in purified human mTECs. Both antigens are expressed in 1% to 3% of mTECs, either individually or coexpressed in the same cell. Using a panel of anti-MUC1 monoclonal antibodies recognizing different post-translational modifications, i.e., glycoforms of MUC1, we show that only fully glycosylated forms of MUC1 and the differentiation-dependent glycoforms were detected on mTECs, but not the cancer-associated glycoforms. Our findings imply that MUC1 and CEA are amenable to central tolerance induction, which might, however, be incomplete in case of tumor cell-restricted MUC1 glycoforms. Knowledge of these subtleties in promiscuous gene expression may, in the future, assist the selection of T cell tumor vaccines for clinical trials.

Intracellular localization of ornithine decarboxylase and its regulatory protein, antizyme-1.

The enzyme ornithine decarboxylase (ODC) and its regulatory protein antizyme-1 (AZ1) are key regulators in the homeostasis of polyamines. To gain more insight into the exact intracellular distribution of ODC and AZ1, we performed immunocytochemical and Green Fluorescent Protein-fluorocytochemical studies in cultured human cervix carcinoma and human prostatic carcinoma (PC-346C) cells. ODC localization patterns varied from predominantly cytoplasmic to both cytoplasmic and nuclear staining, whereas AZ1 was mostly found in the nucleus. In cells that were synchronized in the mitotic phase, localization of both ODC and AZ1 changed from perinuclear at the beginning of mitosis into nucleoplasmic at close proximity to the chromosomes during meta-, ana- and telophase. Upon completion of mitosis, localization of ODC and AZ1 was reverted back to the cytoplasm, i.e., predominantly perinuclear immediately after cytokinesis. When PC-346C cells were treated with polyamines to induce AZ1-regulated ODC degradation, ODC was predominantly found in the nucleus and colocalized with immunoreactive AZ1. A comparable accumulation of ODC and AZ1 in the nucleus was found in PC-346C cells treated with the polyamine analog SL-11093. The present study suggests that AZ1 is involved in nucleocytoplasmic shuttling of ODC, which may be a prerequisite for ODC regulation and/or function.

Mucin-1 is expressed on dendritic cells, both in vitro and in vivo.

Dendritic cells (DCs) are the best professional antigen-presenting cells to stimulate cytotoxic as well as T helper cells and are therefore appropriate candidates for establishing immunotherapy. The concept of our vaccination program is to introduce the tumor-associated antigen mucin-1 (MUC1) into DCs. Analysis of immature and mature DCs--before transducing the antigen MUC1--already demonstrated expression of MUC1 on in vitro monocyte-derived DCs upon maturation. Different culture methods as well as maturation cocktails showed similar results concerning the upregulation of MUC1 expression. Furthermore, we studied the expression of MUC1 on DCs in vivo. No MUC1 expression was found on blood DCs, or on thymic or tonsil DCs. On the other hand, synovial fluid from patients with arthritis contained DCs that were found to express MUC1. This study shows for the first time that the tumor-associated antigen MUC1 is expressed on in vivo DCs. We further show that MUC1 is also expressed on in vitro cultured bone marrow-derived DCs of human MUC1 transgenic mice, supporting the relevance of this mouse model to the human situation. The observation that MUC1 is present on in vivo DCs suggests a functional role, but this physiological function remains to be elucidated.