Potential predictive biomarkers of adalimumab response in patients with hidradenitis suppurativa.

BACKGROUND: Adalimumab provides significant efficacy for patients with hidradenitis suppurativa (HS), which was demonstrated by at least 50% of patients achieving a clinical response by week 12 that was maintained through to week 168 in the PIONEER trials. OBJECTIVES: To identify whether there are biomarkers that could predict adalimumab response, as well as markers that differentially respond to adalimumab in patients with HS. METHODS: Baseline and week-12 plasma samples from the PIONEER studies were used to assess the levels of circulating proteins by multiplex and enzyme-linked immunosorbent assays. RESULTS: Analyses revealed significantly higher high-sensitivity C-reactive protein (hs-CRP) and chemokine (C-C motif) ligand (CCL) 16 (HCC-4) levels in nonresponders at baseline and identified a multivariate response signature of calprotectin, fractalkine and HCC-4, reaching an 86% predictive accuracy rate for adalimumab response. Additionally, post-treatment reduction of plasma C-X-C motif chemokine ligand (CXCL)9, CXCL8 (interleukin-8) and CCL19 (macrophage inflammatory protein 3ß) were greater in adalimumab super-responders than in nonresponders (P = 0·026, P = 0·044 and P = 0·026, respectively). These cytokines are involved in the recruitment of innate and adaptive inflammatory cells, and/or stimulation of certain inflammatory responses, suggesting that these pathways could be disease drivers in adalimumab nonresponders. CONCLUSIONS: These initial results suggest HCC-4, calprotectin and fractalkine could be potential predictive biomarkers of adalimumab response in HS and identified possible tumour necrosis factor-independent disease pathways.

NK cell functions restrain T cell responses during viral infections.

NK cell functions for regulation of T cell responses were evaluated during acute viral infections. In vivo depletion studies established that the presence of NK cells in murine cytomegalovirus (MCMV)-infected immunocompetent mice negatively affected CD4 and CD8 T cell IFN-gamma expression, bromodeoxyuridine (BrdU) incorporation, and expansion. To evaluate NK cell effects, under conditions when NK cells do not control viral replication, experiments were performed using lymphocytic choriomeningitis virus (LCMV). Depletion of NK cells did not affect LCMV-elicited T cell responses in immunocompetent mice; however, the presence of NK cells did inhibit CD4 T cell IFN-gamma production, BrdU incorporation, and expansion in infected MHC class I- and CD8 T cell-deficient beta2M-/- mice. Together, the results reveal a previously unappreciated immunoregulatory role of NK cells for downstream T cell responses.

Replication of murine cytomegalovirus in differentiated macrophages as a determinant of viral pathogenesis.

Blood monocytes or tissue macrophages play a pivotal role in the pathogenesis of murine cytomegalovirus (MCMV) infection, providing functions beneficial to both the virus and the host. In vitro and in vivo studies have indicated that differentiated macrophages support MCMV replication, are target cells for MCMV infection within tissues, and harbor latent MCMV DNA. However, this cell type presumably initiates early, antiviral immune responses as well. In addressing this paradoxical role of macrophages, we provide evidence that the proficiency of MCMV replication in macrophages positively correlates with virulence in vivo. An MCMV mutant from which the open reading frames M139, M140, and M141 had been deleted (RV10) was defective in its ability to replicate in macrophages in vitro and was highly attenuated for growth in vivo. However, depletion of splenic macrophages significantly enhanced, rather than deterred, replication of both wild-type (WT) virus and RV10 in the spleen. The ability of RV10 to replicate in intact or macrophage-depleted spleens was independent of cytokine production, as this mutant virus was a poor inducer of cytokines compared to WT virus in both intact organs and macrophage-depleted organs. Macrophages were, however, a major contributor to the production of tumor necrosis factor alpha and gamma interferon in response to WT virus infection. Thus, the data indicate that tissue macrophages serve a net protective role and may function as "filters" in protecting other highly permissive cell types from MCMV infection. The magnitude of virus replication in tissue macrophages may dictate the amount of virus accessible to the other cells. Concomitantly, infection of this cell type initiates the production of antiviral immune responses to guarantee efficient clearance of acute MCMV infection.

Generation of antigen-specific cytotoxic T lymphocytes and regulation of cytokine production takes place in the absence of CD3zeta.

The TCR-associated protein CD3zeta plays a major role in regulating the state of responsiveness to peptide-MHC complexes on the surface of antigen-presenting cells. In this paper the requirement of CD3zeta in the generation of cytotoxic T cells was compared with its requirement in cytokine gene activation in two mutant mice: ZKO mice with a disrupted CD3zeta gene and ZTG mice in which a truncated CD3zeta segment was expressed as a transgene on the ZKO background. Upon infection of ZTG mice with lymphocytic choriomeningitis virus (LCMV), antigen-specific cytotoxic T lymphocyte (CTL) responses were detected, identical to responses in wild-type mice. In addition, antigen-specific CTL responses to allogeneic class I and class II MHC in ZTG animals were indistinguishable from those in wild-type animals. However, CTL responses to the same major antigens were not detectable in ZKO mice. We conclude that the signal transduction pathways leading to CTL development and cytokine production can be triggered through TCR in the absence of functional CD3zeta, provided the remainder of the TCR-CD3 complex is expressed at high levels on the cell surface. Surprisingly, IFN-gamma production in response to LCMV followed the same kinetics in ZKO, ZTG and wild-type mice. However, in vitro studies showed that cytokine production in general was abnormally regulated in T lymphocytes from ZKO mice, in contrast to ZTG T cells. Taken together, these studies support the hypothesis that development of CTL can take place in the absence of functional CD3zeta. However, CTL development requires stronger TCR-initiated signal transduction events than induction of cytokine genes.

Endogenous glucocorticoids protect against cytokine-mediated lethality during viral infection.

Certain cytokines activate the hypothalamic-pituitary-adrenal axis for glucocorticoid release, and these hormones can protect against cytokine-mediated pathologies. However, endogenous activation of such a pathway has not been established during infections. A prominent glucocorticoid response peaks 36 h following murine CMV (MCMV) infection, coincident with circulating levels of the cytokines IL-12, IFN-gamma, TNF, and IL-6, and dependent on IL-6 for maximal release. These studies examined functions of the hormone induction. Mice rendered glucocorticoid deficient by adrenalectomy were more susceptible than intact mice to MCMV-induced lethality, and the increased sensitivity was reversed by hormone replacement. Lack of endogenous glucocorticoids resulted in increases in IL-12, IFN-gamma, TNF, and IL-6 production, as well as in mRNA expression for a wider range of cytokines, also including IL-1 alpha and IL-1 beta. Viral burdens did not increase, and actually decreased, in the livers of glucocorticoid-deficient mice. TNF, but not IFN-gamma, was required for increased lethality in the absence of endogenous hormone. These results conclusively demonstrate the importance of induced endogenous glucocorticoids in protection against life-threatening effects resulting from infection-elicited cytokine responses. Taken together with the dependence on induced IL-6, they document existence of an immune system-hypothalamic-pituitary-adrenal axis pathway for regulating endogenous responses to viral infections.

Characterization of early cytokine responses and an interleukin (IL)-6-dependent pathway of endogenous glucocorticoid induction during murine cytomegalovirus infection.

Early infection with murine cytomegalovirus (MCMV) induces circulating levels of interleukin (IL)-12, interferon (IFN)-gamma, and tumor necrosis factor (TNF). Studies presented here further characterize these responses by defining kinetics and extending evaluation to include IL-1, IL-6, and glucocorticoids. IL-12 p40, IFN-gamma, TNF, IL-1alpha, and IL-6 were shown to be increased, but IL-1beta was undetectable, in serum of MCMV-infected mice. The IL-12 p40, IFN-gamma, TNF, and IL-6 responses were dramatic with peak levels reaching >150-10,000 pg/ml at 32-40 h after infection and rapidly declining thereafter. Glucocorticoid induction, peaking at 36 h and reaching 30-fold increases above control values, accompanied the cytokine responses. Mice with cytokine deficiencies or neutralized cytokine function demonstrated that IL-6 was the pivotal mediator of the glucocorticoid response, with IL-1 contributing to IL-6 production. The IL-6 requirement appeared to be specific for virus-type stimuli as the synthetic analogue of viral nucleic acid, polyinosinic-polycytidylic acid, also induced IL-6-dependent glucocorticoid release, but treatments with the bacterial product lipopolysaccharide and a non-immune physical restraint stressor elicited IL-6-independent responses. Collectively, the results identify IL-6 as a primary mediator of glucocorticoid induction, and elucidate specific pathways of interactions between immune and neuroendocrine systems during viral infection.

Plasma IgE levels, activation marker expression, and cytokine production in non-atopic individuals.

BACKGROUND: Since human IgE serum levels are very low compared with the other immunoglobulin isotypes, most studies have examined the regulation of IgE production in severely atopic individuals where serum IgE levels are increased. Since atopy is a pathologic consequence of increased IgE production, this disease state could have other influences that result in abnormal expression of these parameters and may not reflect normal IgE regulatory mechanisms. OBJECTIVE: To study nonatopic individuals to examine the expression of IgE regulatory cytokines as well as additional cell surface activation markers in relation to serum IgE levels. METHOD: We selected ten individuals at both the lower and higher end of a spectrum of plasma IgE concentrations from 29 nonatopic individuals and compared the differences in cytokine and activation marker expression in relation to IgE production. RESULTS: We found that even upon extensive examination of activation markers on T, B, and NK cell subsets, there are no significant differences in the cell populations or surface marker expression between the high IgE and low IgE groups. Messenger RNA expression in peripheral blood mononuclear cells (PBMCs) of the cytokines IL-4 and IL-6 was significantly higher, whereas IL-10 was lower in the high IgE group. In addition upon in vitro polyclonal stimulation of peripheral blood mononuclear cells, individuals of the high IgE group produced lower levels of IL-2, IFN gamma and IL-10 compared to the low IgE donors. CONCLUSION: Since our results differ from studies using atopic individuals, this study demonstrates the importance of using nonatopic individuals for examining associations between various immune parameters and IgE.

Decreased production of IL-2 and IFN-gamma by stimulated splenocytes from mice bearing plasma cell tumors is associated with alteration of DNA-binding factors.

We have previously demonstrated that polyclonally stimulated splenocytes as well as enriched T cells from mice bearing plasma cell tumors (PCT) show decreased production of the Th1-associated cytokines, IL-2 and IFN-gamma. This observed loss of IL-2 and IFN-gamma production could be attributed to possible alterations in various factors required for T cell activation and cytokine production. We find that B7 co-stimulatory molecules and IL-2R are up-regulated normally on splenocytes from PCT mice. Concanavalin A (Con A) stimulation of splenocytes from PCT mice in the presence of immobilized anti-CD28 antibody does not enhance proliferation. Exogenous rIL-2 addition to cultures of splenocytes from PCT mice also does not enhance proliferative responses or cytokine production. Furthermore, we do not observe inhibition of normal splenocyte proliferation and IL-2 production in the presence of splenocytes from PCT mice, suggesting that the appearance of suppressor cells cannot account for the decreased responses by splenocytes from PCT mice. Also, IL-2 mRNA levels are decreased in stimulated splenocytes from PCT mice, suggesting that there may be an alteration of transcription factors required for activation of IL-2. Therefore, we have evaluated the DNA-binding activity of transcription factors involved in activation of IL-2 and IFN-gamma gene transcription. We find that binding activities of AP-1, Oct-1 and Oct-2 transcription factors in stimulated splenocytes from PCT mice are similar to normal splenocytes. However, the binding activities of NF-kappa B complexes and factors that bind to the proximal conserved element in the IFN-gamma promoter are dramatically altered in splenocytes from PCT mice. These results suggest that PCT induce changes in certain transcription factors that are important for anti-tumor responses, including T cell proliferative responses and Th1-associated cytokine production.

A combination of IL-10 and direct contact with plasma cell tumors decreases CD23 expression on splenic B cells.

We and others have previously found that splenic B cells from plasma cell tumor-bearing mice exhibit decreased CD23 expression. In the present study we further examined the mechanism of CD23 down-regulation by plasma cell tumors. We show here that although direct contact is required between the tumor cells and B cells, it is not sufficient, since fixed tumor cells do not induce the same reduction in CD23 expression. We have identified IL-10, a cytokine produce by the tumors, as the sole soluble factor that contributes to decreased CD23 expression on B cells induced by plasma cell tumors because 1) Abs to IL-10 prevent the loss of CD23 induced by plasma cell tumors both in vitro and in vivo; 2) engineered IL-10 negative variants of these tumors are reduced in their ability to down-regulate CD23 expression; 3) rIL-10 alone induces partial, but significant, decreases in CD23 expression on normal splenic B cells; and 4) the addition of IL-10 and fixed tumor cells to cultures of normal splenocytes decreases CD23 expression to levels similar to those in cocultures with live tumor cells. Collectively, these results demonstrate that plasma cell tumors down-regulate CD23 expression on B cells by a coordinate mechanism of IL-10 plus contact-mediated events and reveal a novel role for IL-10 in the regulation of CD23 expression on B cells that is suggestive of host B cell activation in the presence of the tumor.

In vitro and in vivo recovery of IFN-gamma, but not IL-2, production by IL-12 in mice with plasma cell tumors.

We have previously found that T cells from mice bearing plasma cell tumors (PCT mice) demonstrate decreased proliferation as well as decreased production of the Th 1-associated cytokines IL-2 and IFN-gamma in response to polyclonal stimulation. In the present study, we have examined soluble factors as possible elements required to rescue this decreased proliferation and cytokine production by splenocytes from PCT mice. We find that the addition of supernatants from stimulated normal splenocytes has no effect on proliferation of IL-2 production by splenocytes from PCT mice. In contrast, these supernatants completely restore IFN-gamma production by splenocytes from PCT mice. We have found that IL-12 is responsible for the observed increase in IFN-gamma production because: (i) addition of anti-IL-12 antibody blocks this recovery of IFN-gamma production by these supernatants, (ii) the addition of recombinant IL-12 to cultures of splenocytes from PCT mice results in increased IFN-gamma production and (iii) in vivo treatment of PCT mice in IL-12 also results in increased IFN-gamma production by the subsequently activated splenocytes, but has little effect on proliferation or IL-2 production. These results demonstrate that both in vitro and in vivo, IL-12 selectively restores the decreased production of IFN-gamma by splenocytes from PCT mice.

Plasma cells tumors decrease CD23 mRNA expression in vivo in murine splenic B cells.

CD23, the low-affinity Fc receptor for IgE, is constitutively expressed on mature, naive B cells, but is lost following B cell activation. We and others have shown that CD23 expression on B cells decreases in mice bearing plasma cell tumors. In contrast to these findings, we find that IgE-secreting tumors do not cause a loss of CD23 expression on host splenic B cells. Decreased expression of CD23 on B cells induced by plasma cell tumors requires direct contact between tumor cells and B cells; and other host cells are not involved. The loss of surface CD23 expression is associated with a decrease in steady-state CD23 mRNA levels in B cells from plasma cell tumor-bearing mice. Interestingly, loss of CD23 mRNA is observed even in B cells from mice with IgE-secreting tumors, where we find surface CD23 protein expression to be similar to that of normal mice. The maintenance of surface CD23 expression on B cells in mice with IgE-secreting tumors is dependent solely on the presence of IgE, and is not a tumor-specific effect. Therefore, we conclude that in vivo, IgE secreted by plasma cell tumors can result in the maintenance of normal levels of surface CD23 expression by a post-transcriptional mechanism, even when the tumor induces a down-regulation of CD23 mRNA levels.

Specific decrease of Th1-like activity in mice with plasma cell tumors.

Previously we examined the ability of the host's immune responses to regulate Ig production in an IgE-secreting murine plasma cell tumor (B53). In the present study we have examined the reverse phenomenon, in that we have investigated the effects of this and other plasma cell tumors on the immune responses of their hosts. We found that splenocytes from plasma cell tumor-bearing mice demonstrate decreased proliferation in response to polyclonal stimulation by either Con A or a combination of PMA and calcium ionophore (A23187). Fractionation of the splenocytes demonstrated that this reduction in proliferation was confined to CD4+ T cells and that the proliferation of CD8+ T cells was unaffected. In order to determine whether the down-modulatory effects of the tumor were confined to a particular CD4+ helper T cell subset, we examined the production of cytokines representing the Th1 subset (IL-2 and IFN-gamma) and the Th2 subset (IL-4 and IL-10) from stimulated splenocytes and from stimulated enriched splenic T cells. We found that both stimulated splenocytes and T cells from plasma cell tumor-bearing mice produced lower levels of the Th1 cytokines IL-2 and IFN-gamma compared with normal cultures, demonstrating that Th1-like responses are inhibited in the hosts of these tumors. However, no alterations in the production of the Th2 cytokines IL-4 and IL-10 were observed in these stimulated splenocyte or T cell cultures from the tumor-bearing mice.(ABSTRACT TRUNCATED AT 250 WORDS)

Metabolism of 2-aminofluorene by human polymorphonuclear leukocytes: more evidence for the association between inflammation and cancer.

Recent investigations have demonstrated the ability of leukocytes to metabolize promutagens or procarcinogens into their genotoxic forms. As a possible explanation for the association between inflammation and cancer, we and others have hypothesized that local accumulations of leukocytes could take up nearby promutagens, metabolize them, and release genotoxic agents that may cause damage in the surrounding tissue. Using a modified, two-step preincubation protocol with Salmonella, we have tested this hypothesis. We have shown that total human peripheral blood leukocytes, cultured in the presence of 2-aminofluorene for 18 hr, can metabolize 2-aminofluorene into agents mutagenic to Salmonella typhimurium strain TA98. Furthermore, experiments in which polymorphonuclear leukocytes were separated from mononuclear leukocytes demonstrated that the PMNs metabolized 2-aminofluorene to a much greater extent than the MNs.