Immunomodulatory effects of sucupira (Pterodon pubescens) seed infusion on collagen-induced arthritis.

OBJECTIVE: To evaluate the clinical and immunomodulatory efficacy of seed extracts from sucupira branca (Pterodon pubescens Benth.), a Brazilian anti-inflammatory folk medicine, against collagen II (CII)-induced arthritis (CIA) in mice. METHODS: Mice were divided into 3 groups: 1) normal control mice received a vehicle (ethanol 15% in water); 2) mice with CIA received the vehicle; and 3) mice with CIA received extract from 1 mg sucupira seeds/day. The daily oral treatments started 21 days after the first collagen immunization, ending 4 weeks later. We analyzed the arthritic index, the histopathology of the joints, the serum anti-CII IgG antibody level, and the absolute counts of the CD4+, CD8+, CD4+CD69+ and CD8+CD69+ subsets of inguinal lymph nodes (LN). RESULTS: Sucupira treatment strongly reduced the severity of arthritis (p < 0.001). Vehicle-treated CIA mice exhibited invasive synovial pannus and significant articular leukocyte infiltration, features that were reduced or absent in sucupira-treated mice. Mice with CIA exhibited twice the number of CD4+ and CD8+ LN cells found in control mice. Suctupira-treated mice exhibited these subsets in numbers comparable to the latter. A two-thirds decrease in the level of serum anti-CII IgG antibody and a normalization of the CD4+CD69+ LN cell number in treated mice hallmark a negative regulatory effect of sucupira on B- and CD4 T-cell activation, respectively. The CD8+CD69+ cell number remained roughly the same in the 3 groups. CONCLUSION: The clinical and immunomodulatory effects of sucupira on CIA provides a further experimental basis for the popular use of sucupira in chronic inflammatory diseases.

Acute and topic anti-edematogenic fractions isolated from the seeds of Pterodon pubescens.

We previously demonstrated that alcoholic extracts from Pterodon pubescens Benth. (Sucupira branca, Leguminosae) seeds exhibit anti-arthritic activity. In the present work we show that the oleaginous extract obtained from P. pubescens seeds (OEP) exhibits acute or topic anti-edematogenic activity when tested in carrageenan-induced paw edema or in croton oil-induced ear edema assays, respectively. Four fractions were obtained from OEP by sequential liquid-liquid extraction. The anti-edematogenic properties were predominant in the hexanic fraction, which was further fractionated by HPLC, yielding three sub-fractions (PF1.1, PF1.2 and PF1.3). PF1.1 and PF1.3 showed potent acute and topic anti-edematogenic activity. The PF1.2 sub-fraction, although not active in the carrageenan assay, exhibited a potent anti-edematogenic activity in the croton oil-induced ear edema. This sub-fraction shows a maximum efficacy similar to indometacin in a lower dose. The PF1.1 sub-fraction presented a complex mixture containing furane diterpene derivatives of vouacapan. PF1.2 consists of a single substance, geranylgeraniol, as determined by GC/MS and NMR, while PF1.3 contains farnesol.

The "early' CD4+CD8+ stage of thymocte differentiation hallmarks the end of a strong positive correlation between extracellular matrix receptor expression and protein tyrosine phosphorylation.

We used irradiation-induced thymic regression/reconstitution to study phosphotyrosine (PTyr) levels and expression of extracellular matrix receptors in thymocyte subsets by flow cytometry. High PTyr levels (PTyr(hi)) characterized cells from the CD4-CD8-(DN)CD25in/hi to the "early" CD4-CD8+(DP)CD25- stage. Correlation indexes (R) between the percentages of these PTyrhi cells and cells with up-regulated expression of alpha4 integrin (alpha4hi) were strongly positive (R= 0.91, P= 0.002, for DN; R= 0.98, P= 0.0001 for DP). At the "early" DP stage, R between PTyrhi cells and cells with up-regulated expression of alpha5 integrin and L-selectin (alpha5hi and L-sel(hi)) also rendered strongly positive (R>0.95, p<0.0003). "Late" expanding DP cells exhibited intermediate PTyr levels (PTyr(in)), associated with a down-regulation of the adhesion receptors assessed. Triple-labeling suggested that in most early CD3-/lo cells, alpha4hi and alpha5hi, but not L-sel(hi) expression preceded a PTyr(hi) content. CD3in/hi-enriched CD8+ cells were also PTyr(hi), but conversely to the immature ones exhibited a tendency for a negative R between PTyr(hi) and alpha4hi (R = -0.93, P = 0.067, n= 4) or alpha5hi cells (R = -0.77, P = 0.23, n = 4). CD4+ cells were either PTyr(hi) or PTyr(in), exhibiting a tendency for a positive R (R = 0.59, P = 0.124, n= 8) between PTyr(hi) and L-sel(hi) cells only. In conclusion, our results associate an up-regulation of alpha4 and alpha5 chains expression with PTyr(hi) levels and, as elsewhere published, with increased adhesion to fibronectin up to the "early" DP stage, but not afterwards.

Role of extracellular matrix-mediated interactions in thymocyte migration.

Cell adhesion, migration, differentiation and survival or death is amongst a large spectrum of biological responses that can be elicited by ligation of extracellular matrix components to their corresponding receptors. As regards the physiology of the thymus, cell migration is a crucial event in the general process of T cell differentiation. Studies on the intrathymic distribution of ECM components revealed that fibronectin, laminin and type IV collagen, are not restrictedly located at typical basement membrane sites, also forming a thick network in the medullary region of the thymic lobules, whereas very thin ECM fibers are found within the cortex. These ECM components are essentially produced by thymic microenvironmental cells, which also drive thymocyte differentiation. Signals triggered by ECM are conveyed into thymocytes or microenvironmental cells through specific membrane receptors, and most of them belong to the integrin type, such as the VLA-3, VLA-4, VLA-5 and VLA-6. In vitro studies revealed that adhesion of thymocytes to thymic microenvironmental cells is mediated by extracellular matrix. Such an adhesion is preferentially done by immature thymocytes. Importantly, ECM-mediated interactions also govern the entrance and exit of thymocytes in the lymphoepithelial complexes named thymic nurse cells. Lastly, pathological conditions, including infectious and autoimmune diseases, in which changes of ECM ligands and receptors are observed, course with alterations in thymocyte migration and death. In conclusion, the fact that ECM can modulate traffic, differentiation, death and survival of normal thymocytes adds clues for understanding how ECM-mediated interactions behave in the thymus, not only in normal, but also in pathological conditions.

Successful treatment of collagen-induced arthritis in mice with a hydroalcohol extract of seeds of Pterodon pubescens.

The antiarthritic effect of a hydroalcohol extract of Pterodon pubescens (HEPp) seeds was tested using collagen-induced arthritis (CIA) in DBA1/J mice treated with daily oral doses of HEPp in different schedules. The preventive treatment significantly reduced both the arthritic index (AI) and the CIA incidence. Using a therapeutic protocol, only the lower dose of HEPp induced a decrease in both parameters. These results provide a scientific foundation for the popular use of Pp seed infusions in rheumatoid arthritis (RA) treatment.

Upregulated expression of fibronectin receptors underlines the adhesive capability of thymocytes to thymic epithelial cells during the early stages of differentiation: lessons from sublethally irradiated mice.

A 250-cGy whole-body gamma-radiation dose was used to induce thymus regression in mice, and to study the expression and function of extracellular matrix (ECM) receptors in distinct thymocyte subsets emerging during repopulation of the organ. The onset of regeneration was detected from day 2 to 3 postirradiation (P-Ir), when a remarkable increase in the absolute counts of CD3(-)CD25(hi)CD44(+) and CD3(-)CD25(in/hi)CD44(-) cells occurred. Enhanced expression of L-selectin, alpha4, and alpha5 integrin chains (L-selhi alpha4(hi) alpha5(hi)) was also exhibited by these cells. This pattern of expression was maintained until the CD4(+)CD8(+) (DP) young stage was achieved. Afterward, there was a general downregulation of these ECM receptors in DP as well as in CD4(+) or CD8(+) single positive (SP) thymocytes (L-selin alpha4(in) alpha5(in)). In some recently generated SP cells, alpha4 expression was downregulated before the alpha5 chain, and L-selectin was upregulated in half of more mature cells. The expression of the alpha6 integrin chain was downregulated only in maturing CD4(+) cells. Importantly, the increased expression of L-selectin and alpha4 and alpha5 chains in thymocytes was strongly correlated with their adhesiveness to thymic epithelial cells (TEC) in vitro. Blocking experiments with monoclonal antibody or peptides showed the following: (1) that the LDV rather than the REDV cell attachment motif in the IIIC segment of fibronectin is targeted by the alpha4 integrin during thymocyte/TEC adhesion; (2) that the RGD motif of the 120-kD fragment of fibronectin, a target for alpha5 integrin, has a secondary role in this adhesion; and (3) that the YIGSR cell attachment motif of the beta1 chain of laminin/merosin recognized by a nonintegrin receptor is not used for thymocyte adherence. In conclusion, our results show that an upregulated set of receptors endows CD25(+) precursors and cells up to the young DP stage with a high capability of interacting with thymic ECM components.

Epidermal growth factor modulates fetal thymocyte growth and differentiation.

In the present study, we used the fetal organ culture (FTOC) technique in order to study a putative effect of epidermal growth factor (EGF) on the thymus ontogeny. Functional EGF receptors and more recently the EGF molecule itself, respectively, on the membrane of epithelial components of thymic stroma and on a few thymocytes in adult thymus, had been reported in the literature. We could observe a dose-dependent decrease in cellularity and a progressive retention of thymocytes in the double-negative (CD4-/CD8-) stage of differentiation when exogenous EGF was added. Epidermal growth factor interfered with both fetal stroma growth and thymocyte development at a precise moment, that is, in the passage from double-negative to the double-positive (CD4+/CD8+) stage. After a 7-day FTOC in the presence of EGF, most cells recovered were Thy-1.2+, c-kit+, TSA1-/int, CD3-, and one of CD44high/CD25int, CD44-/CD25int, or CD44/CD25-. Some developed into gammadeltaTCR+ cells with a mature (CD3+) phenotype, but not into alphabetaTCR+ thymocytes. It seems that EGF addition makes the cultures "nonpermissible" for alphabetaTCR+ thymocyte generation. We report here the presence of a high Mr "EGF-like" molecule on the membrane of fetal thymocytes, which role in the observed effects is under investigation. Further biochemical characterization of this molecule is still required, because its presence was only evidenced on the basis of its antigenicity.

Epidermal growth factor (EGF) modulates fetal thymocyte growth and differentiation: partial reversal by insulin, mimicking by specific inhibitors of EGF receptor tyrosine kinase activity, and differential expression of CD45 phosphatase isotypes.

We have recently reported that epidermal growth factor (EGF) modulates thymocyte development in fetal thymus organ cultures. Exogenously added EGF arrested thymocyte growth and differentiation, acting at the transition from the CD4-CD8- (double-negative (DN)) to the CD4+CD8+ (double-positive (DP)) phenotype. In this study, we further investigate some molecular aspects of this blockade. This inhibitory effect could be mimicked by tyrphostins, which are selective inhibitors of EGF receptor kinase activity. An attempt to use insulin (INS) as a synergizing effector resulted in partial restoration of lobe cellularity, leading to expansion of the CD44-CD25+ DN subset. However, INS did not overcome the EGF-driven blockade of the thymocyte DN --> DP transition. Analysis of CD45 phosphatase showed that this transition was preceded by a rise in CD45RB isotype expression. At the end of a 7-day culture, the remaining DN cells from both EGF- and EGF+INS-treated fetal thymus organ cultures showed a CD45RB- phenotype and were negative for the EGF-immunoreactive molecule described previously on the fetal thymocyte surface. This finding implies that neither molecule is related to the growth capability of cells at this early developmental stage; it is more likely that the molecules are related to subsequent events in the thymocyte pathway to the DP phenotype. Thus, our data support the concept that EGF receptor-related circuitry may be relevant in thymus ontogeny. Additionally, evidence is provided for the duality between growth and differentiation at this particular early stage of thymocyte development.

Radio- and chemoprotection of bone marrow cells by opposite cell cycle-acting cytokines.

Cytokines such as interleukin-1 (IL-1), tumor necrosis factor alpha (TNF-alpha), stem cell factor (SCF), and interleukin-12 (IL-12), among others, are presently known to exert a radioprotective effect on bone marrow (BM) precursor cells. IL-1, TNF-alpha, transforming growth factor-beta (TGF-beta), and macrophage inflammatory protein-1 alpha (MIP-1alpha) exert a chemoprotective effect on BM cells, while a putative role of IL-12 in this effect is still unknown. IL-1, SCF, and IL-12 are known to promote BM precursor cell cycling. Conversely, TNF-alpha, MIP-1alpha, and TGF-beta, the latter a radiosensitizer, induce cycle arrest in these cells. Cycling increases radioprotection, while arrest reduces chemical damage. IL-1 and TNF-alpha are unique in their ability to induce detoxifying mechanisms. The present communication overviews these effects. It also proposes a model, based on the induction of biochemical detoxifying mechanisms, aiming to explain BM cell radio- and chemoprotection by opposite cell cycle-acting cytokines.

In vivo and in vitro expression of tenascin by human thymic microenvironmental cells.

Increasing evidence reveals that extracellular matrix components can be regarded as a group of mediators in intrathymic T-cell migration and/or differentiation. Yet, little is known about the expression and putative function of one particular extracellular matrix protein, namely, tenascin in the thymus. Herein we investigated, by means of immunocytochemistry, tenascin expression in normal infant and fetal human thymuses, as well as in cultures of thymic microenvironmental cells. In situ, tenascin distribution is restricted to the medulla and cortico-medullary regions of normal thymuses. This pattern thus differed from that of fibronectin, laminin and type IV collagen, in which subseptal basement membranes were strongly labeled. Interestingly, tenascin did not co-localize with the cytokeratin-defined thymic epithelial cell network. This was in keeping with the in vitro data showing that tenascin-bearing cells were nonepithelial (and probably nonfibroblastic) microenvironmental elements. Studies with fetal thymuses revealed a developmentally regulated expression of tenascin, with a faint but consistent network labeling, in thymic rudiments as early as 12 weeks of gestational age, that progressed to a strong TN expression at 18 weeks of fetal development, which was similar to the distribution pattern observed thereafter, including postnatally. Our results clearly indicated that tenascin is constitutively expressed in the human thymus, since early stages of thymic ontogeny, and suggest that the cell type responsible for its secretion is a nonepithelial microenvironmental cell.

Interleukin-1 and tumor necrosis factor-alpha as radio- and chemoprotectors of bone marrow.

Administration of interleukin 1 (IL-1) or tumor necrosis factor-alpha (TNF alpha) protects bone marrow precursor cells (BMPC) from ionizing radiation and antineoplastic drugs. The time of injection is critical: the best protective results being obtained when cytokines are given around 24h prior to the induced injury. Multiple daily cytokine injections that precede irradiation or drug administration are more effective than single ones although single doses are quite effective at increasing survival in mice. Protection is positively correlated with both rapid granulocyte recovery and BMPC survival. Mechanisms involved in BMPC radioprotection include: (1) push to the S/G2 + M or arrest in the G0 phases of the cell cycle by IL-1 or TNF alpha, respectively, and (2) induction of mitochondrial manganous superoxide dismutase synthesis. For BMPC chemoprotection, proposed mechanisms are: (1) increase of aldehyde dehydrogenase synthesis, and (2) modulation of multiple-drug resistant gene expression. Stimulation of glutathione synthesis in BMPC could be operating in both radio- and chemoprotection. These findings point to the relevance of IL-1 or TNF alpha in cancer therapy as a means of reducing BMPC sensitivity to cytoreductive drugs or irradiation (including radioimmunotherapy) as well as in in vitro tumor cell purging with drugs in autologous BMT. Prior administration of these cytokines should be also considered for people in imminent danger of exposure to radiation.

A modified assay for measuring thymocyte co-stimulatory activity.

The observation that murine thymocytes increase their proliferation to interleukin 1 (IL-1) in the presence of phytohemagglutinin (PHA) when pre-incubated with interleukin 2 (IL-2) allowed the introduction of a modified assay for the measurement of IL-1 or the search of thymocyte-inducing proliferative activities in biological samples. Pre-incubation of thymocytes for 24 hr with 50 u/ml IL-2, followed by washings, elicited their maximal response to IL-1 in the usual lymphocyte activating factor (LAF) assay. This suggests that sequential events lead to thymocyte activation. The responsiveness is three to five fold greater than, and the total time of assay is the same as that of the LAF assay. Interestingly, pre-incubation with IL-2 renders thymocytes more sensitive than responsive to crude monocyte conditioned media. The use of the MTT colorimetric method for the assessment of thymocyte proliferation, and of the lectin jacalin as a co-mitogen are suggested as alternatives to be used in co-stimulatory assays.

[Hematopoiesis: I. Cytokines involved in its regulation]. / Hematopoese: I. Citocinas envolvidas em sua regulação.

The study of the direct involvement of colony stimulating factors, interleukins, and other purified factors in distinct steps of hematopoiesis has been complicated by the in vitro presence of non-hematopoietic cells which can intermediate the effects observed on hematopoietic precursors. The review covers the recent finding that the CD34 antigen is expressed on the membranes of essentially all pluripotent stem cells, but is lacking in the majority of the differentiated blood and stromal bone marrow cells. This finding allowed in vitro experiments with selected CD34+ hematopoietic precursors, and a consequent reevaluation of the participation of different factors in their differentiation. The role of interferons, tumor necrosis factors, and transforming growth factors beta in the negative regulation of hematopoiesis is also analysed.

Nylon wool column: a tool for obtaining monokine-rich eluates in the absence of serum

Diverse conditions for stimulating human mononuclear cells to release thymocyte costimulatory factors were tested for their contribution to the generation of supernatants high titers of these monokines. Activity titers increased with LPS concentration, reaching a plateau between 1 and 10 microng/ml. Indomethacin did not modify the monokine, but the assay for thymocyte costimulatory activity was substantially affected by inhibitory substances produced by the monocytes in the absence of indomethacin. The use of nylon wool columns to trap the cells was shown to be effective in raising cellular densities without decreasing activity titers. As result, the yield per cell could be maintained even in the absence of serum, an important step toward the goal of purifiying bioactive from crude broths

Nylon wool column--a tool for obtaining monokine-rich eluates in the absence of serum.

Diverse conditions for stimulating human mononuclear cells to release thymocyte costimulatory factors were tested for their contribution to the generation of supernatants containing high titers of these monokines. Activity titers increased with LPS concentration, reaching a plateau between 1 and 10 micrograms/ml. Indomethacin did not modify the monokine release, but the assay for thymocyte costimulatory activity was substantially affected by inhibitory substances produced by the monocytes in the absence of indomethacin. The use of nylon wool columns to trap the cells was shown to be effective in raising cellular densities without decreasing activity titers. As a result, the yield per cell could be maintained even in the absence of serum, an important step toward the goal of purifying bioactive peptides from crude broths.