Regulated production of the chemokine CCL28 in human colon epithelium

The chemokine CCL28 is constitutively expressed by epithelial cells at several mucosal sites and is thought to function as a homeostatic chemoattractant of subpopulations of T cells and IgA B cells and to mediate antimicrobial activity. We report herein on the regulation of CCL28 in human colon epithelium by the proinflammatory cytokine IL-1, bacterial flagellin, and n-butyrate, a product of microbial metabolism. In vivo, CCL28 was markedly increased in the epithelium of pathologically inflamed compared with normal human colon. Human colon and small intestinal xenografts were used to model human intestinal epithelium in vivo. Xenografts constitutively expressed little, if any, CCL28 mRNA or protein. After stimulation with the proinflammatory cytokine IL-1, CCL28 mRNA and protein were significantly increased in the epithelium of colon but not small intestinal xenografts, although both upregulated the expression of another prototypic chemokine, CXCL8, in response to the identical stimulus. In studies of CCL28 regulation using human colon epithelial cell lines, proinflammatory stimuli, including IL-1, bacterial flagellin, and bacterial infection, significantly upregulated CCL28 mRNA expression and protein production. In addition, CCL28 mRNA expression and protein secretion by those cells were significantly increased by the short-chain fatty acid n-butyrate, and IL-1- or flagellin-stimulated upregulation of CCL28 by colon epithelial cells was synergistically increased by pretreatment of cells with n-butyrate. Consistent with its upregulated expression by proinflammatory stimuli, CCL28 mRNA expression was attenuated by pharmacological inhibitors of NF-kappaB activation. These findings indicate that CCL28 functions as an "inflammatory" chemokine in human colon epithelium and suggest the notion that CCL28 may act to counterregulate colonic inflammation.     

宫颈癌中CD1α分子和趋化因子CCL20/MIP-3α的表达

目的:研究CD1α分子,趋化因子人巨噬细胞炎性蛋白-3α(CCL20/MIP-3α)在宫颈癌组织中的表达及其相关性,探讨其在宫颈癌免疫逃逸机制中的作用。方法:应用免疫组化PV-6000通用二步法检测CD1α和趋化因子CCL20/MIP-3α在正常宫颈组织及宫颈癌组织中的表达,并分析两者之间的相关性。结果:CD1α和CCL20/MIP-3α在宫颈癌中的表达均明显降低(P均0.01);CD1α与CCL20/MIP-3α在宫颈癌中的表达显著相关(P0.01)。结论:在宫颈癌组织中,趋化因子的减少导致抗原提呈细胞数量下降,不足以引起肿瘤局部免疫反应而导致了宫颈局部病变的侵袭及发展。     

Production of MDC/CCL22 by human intestinal epithelial cells

The intestinal mucosa contains a subset of lymphocytes that produce Th2 cytokines, yet the signals responsible for the recruitment of these cells are poorly understood. Macrophage-derived chemokine (MDC/CCL22) is a recently described CC chemokine known to chemoattract the Th2 cytokine producing cells that express the receptor CCR4. The studies herein demonstrate the constitutive production of MDC/CCL22 in vivo by human colon epithelium and by epithelium of human intestinal xenografts. MDC/CCL22 mRNA expression and protein secretion was upregulated in colon epithelial cell lines in response to proinflammatory cytokines or infection with enteroinvasive bacteria. Inhibition of nuclear factor (NF)-kappaB activation abolished MDC/CCL22 expression in response to proinflammatory stimuli, demonstrating that MDC/CCL22 is a NF-kappaB target gene. In addition, tumor necrosis factor-alpha-induced MDC/CCL22 secretion was differentially modulated by Th1 and Th2 cytokines. Supernatants from the basal, but not apical, side of polarized epithelial cells induced a MDC/CCL22-dependent chemotaxis of CCR4-positive T cells. These studies demonstrate the constitutive and regulated production by intestinal epithelial cells of a chemokine known to function in the trafficking of T cells that produce anti-inflammatory cytokines.     

CCL3 (MIP-1alpha) induces in vitro migration of GM-CSF-primed human neutrophils via CCR5-dependent activation of ERK 1/2

CCL3 (MIP-1alpha), a prototype of CC chemokines, is a potent chemoattractant toward human neutrophils pre-treated with GM-CSF for 15 min. GM-CSF-treated neutrophils migrate also to the selective CCR5 agonist CCL4 (MIP-1beta). CCL3- and CCL4-triggered migration of GM-CSF-primed neutrophils was inhibited by the CCR5 antagonist TAK-779. Accordingly, freshly isolated neutrophils express CCR5. Extracellular signal-regulated kinases (ERK)-1/2 and p38 mitogen-activated protein kinase (MAPK) inhibitors blocked CCL3-induced migration of GM-CSF-primed neutrophils. When the activation of ERK-1/2 and p38 MAPK by CCL3 and the classical neutrophilic chemokine CXCL8 (IL-8) were compared, both the chemokines were capable of activating p38 MAPK. On the contrary, whereas both ERK-1 and ERK-2 were activated by CXCL8, no ERK-1 band was detectable after CCL3 triggering. Finally, neutrophil pre-treatment with GM-CSF activated both ERK-1 and ERK-2. This suggests that by activating ERK-1, GM-CSF renders neutrophils rapidly responsive to CCL3 stimulation throughout CCR5 which is constitutively expressed on the cell surface.     

Up-regulation of macrophage inflammatory protein-3 alpha/CCL20 and CC chemokine receptor 6 in psoriasis

Autoimmunity plays a key role in the immunopathogenesis of psoriasis; however, little is known about the recruitment of pathogenic cells to skin lesions. We report here that the CC chemokine, macrophage inflammatory protein-3 alpha, recently renamed CCL20, and its receptor CCR6 are markedly up-regulated in psoriasis. CCL20-expressing keratinocytes colocalize with skin-infiltrating T cells in lesional psoriatic skin. PBMCs derived from psoriatic patients show significantly increased CCR6 mRNA levels. Moreover, skin-homing CLA+ memory T cells express high levels of surface CCR6. Furthermore, the expression of CCR6 mRNA is 100- to 1000-fold higher on sorted CLA+ memory T cells than other chemokine receptors, including CXCR1, CXCR2, CXCR3, CCR2, CCR3, and CCR5. In vitro, CCL20 attracted skin-homing CLA+ T cells of both normal and psoriatic donors; however, psoriatic lymphocytes responded to lower concentrations of chemokine and showed higher chemotactic responses. Using ELISA as well as real-time quantitative PCR, we show that cultured primary keratinocytes, dermal fibroblasts, and dermal microvascular endothelial and dendritic cells are major sources of CCL20, and that the expression of this chemokine can be induced by proinflammatory mediators such as TNF-alpha/IL-1 beta, CD40 ligand, IFN-gamma, or IL-17. Taken together, these findings strongly suggest that CCL20/CCR6 may play a role in the recruitment of T cells to lesional psoriatic skin.     

The structure of human macrophage inflammatory protein-3alpha /CCL20. Linking antimicrobial and CC chemokine receptor-6-binding activities with human beta-defensins

Human macrophage inflammatory protein-3alpha (MIP-3alpha; CCL20) is a CC-type chemokine that binds to and activates CC chemokine receptor-6 (CCR6). Although MIP-3alpha does not share the binding site of CCR6 with any other chemokine, human beta-defensin-1 and -2, small cationic antimicrobial peptides, have also been found to bind to and activate CCR6. Conversely, we have found that MIP-3alpha possesses antibacterial activity of greater potency than human beta-defensin-1 and -2 against Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 29213, while having no activity against the fungus Candida albicans. There is no clear sequence similarity between beta-defensins and the chemokine MIP-3alpha, beyond an abundance of cationic residues and the presence of disulfide bonds. Nonetheless, there are structural similarities between these three proteins that allow their overlap of chemotactic and antimicrobial activities. In this report, we describe the x-ray crystal structure of human MIP-3alpha refined to a resolution of 1.7 A and compare it with the crystal structures of human beta-defensin-1 and -2. Molecules of MIP-3alpha and the beta-defensins seem to share few structural motifs that are likely associated with their common biological activities.     

Human macrophage inflammatory protein-3alpha/CCL20/LARC/Exodus/SCYA20 is transcriptionally upregulated by tumor necrosis factor-alpha via a non-standard NF-kappaB site.

The 5'-flanking sequences of the human macrophage inflammatory protein-3alpha/CCL20 gene were cloned and transfected into G-361 human melanoma cells in a luciferase reporter construct. Tumor necrosis factor-alpha (TNF-alpha) treatment stimulated luciferase expression, and promoter truncations demonstrated that TNF-alpha inducibility is conferred by a region between nt -111 and -77, which contains a non-standard nuclear factor-kappaB (NF-kappaB) binding site. The requirement for NF-kappaB was demonstrated as follows: (i) mutations in this NF-kappaB site abrogated TNF-alpha responsiveness; (ii) TNF-alpha activated a construct containing two copies of the CCL20 NF-kappaB binding site; (iii) overexpression of NF-kappaB p65 activated the CCL20 promoter; (iv) NF-kappaB from nuclear extracts of TNF-alpha-stimulated cells bound specifically to this NF-kappaB site.     

Regulated production and molecular diversity of human liver and activation-regulated chemokine/macrophage inflammatory protein-3 alpha from normal and transformed cells

Liver and activation-regulated chemokine (LARC), also designated macrophage inflammatory protein-3alpha (MIP-3alpha), Exodus, or CCL20, is a C-C chemokine that attracts immature dendritic cells and memory T lymphocytes, both expressing CCR6. Depending on the cell type, this chemokine was found to be inducible by cytokines (IL-1beta) and by bacterial, viral, or plant products (including LPS, dsRNA, and PMA) as measured by a specific ELISA. Although coinduced with monocyte chemotactic protein-1 (MCP-1) and IL-8 by dsRNA, measles virus, and IL-1beta in diploid fibroblasts, leukocytes produced LARC/MIP-3alpha only in response to LPS. However, in myelomonocytic THP-1 cells LARC/MIP-3alpha was better induced by phorbol ester, whereas in HEp-2 epidermal carcinoma cells IL-1beta was the superior inducer. The production levels of LARC/MIP-3alpha (1-10 ng/ml) were, on the average, 10- to 100-fold lower than those of IL-8 and MCP-1, but were comparable to those of other less abundantly secreted chemokines. Natural LARC/MIP-3alpha protein isolated from stimulated leukocytes or tumor cell lines showed molecular diversity, in that NH(2)- and COOH-terminally truncated forms were purified and identified by amino acid sequence analysis and mass spectrometry. In contrast to other chemokines, including MCP-1 and IL-8, the natural processing did not affect the calcium-mobilizing capacity of LARC/MIP-3alpha through its receptor CCR6. Furthermore, truncated natural LARC/MIP-3alpha isoforms were equally chemotactic for lymphocytes as intact rLARC/MIP-3alpha. It is concluded that in addition to its role in homeostatic trafficking of leukocytes, LARC/MIP-3alpha can function as an inflammatory chemokine during host defense.     

NMR solution structure of murine CCL20/MIP-3alpha, a chemokine that specifically chemoattracts immature dendritic cells and lymphocytes through its highly specific interaction with the beta-chemokine receptor CCR6

CCL20/MIP-3alpha is a beta-chemokine expressed in the thymus, skin, and intestinal epithelial cells that exclusively binds and activates the CCR6 receptor in both mice and humans. The strict receptor binding specificity of CCL20 is exceptional; other chemokines and their receptors bind promiscuously with multiple partners. Toward determining the structural basis for the selective receptor specificity of CCL20, we have determined its three-dimensional structure by 1H NMR spectroscopy. CCL20 exhibits the same monomeric structure previously described for other chemokines: a three-stranded beta-sheet and an overlying alpha-helix. The CCL20 receptor selectivity could arise from the rigid conformation of the N-terminal DCCL motif as well as the groove between the N-loop and the beta2-beta3 hairpin, which is significantly narrower in CCL20 than in other chemokines. Similar structural features are seen in human beta-defensin 2, a small nonchemokine polypeptide reported to selectively bind and activate CCR6, which stresses their importance for the specific binding of both CCL20 and beta-defensin 2 to CCR6. CCL20's structure will be useful to design tools aimed to modulate its important biological functions.     

Regulation of human eotaxin-3/CCL26 expression: modulation by cytokines and glucocorticoids

Eotaxin-3 (CCL26) is a CC chemokine that signals exclusively via the CCR3 receptor and has eosinophil-selective chemoattractant activity. Comparison of Eotaxin-1 (CCL11) and Eotaxin-2 (CCL24), demonstrates differences in their expression profiles, cell specificity and effector kinetics, implying distinct biological actions. But little data in this regard have been reported for Eotaxin-3. We aimed to analyse the effect of Th2 cytokines and glucocorticoids on Eotaxin-3 mRNA expression in human lung epithelial cells and dermal fibroblasts; cells implicated in the pathogenesis of allergic asthma and allergic dermatitis respectively. Eotaxin-3 mRNA levels in primary dermal fibroblasts and NCI-H727 lung epithelial cells were determined by Northern hybridization. In contrast to Eotaxin-1, Eotaxin-3 mRNA expression was not detected in unstimulated cells. The Th2 cytokines IL-4 and IL-13 induced Eotaxin-3 expression in a time and dose dependent manner, with IL-4 demonstrating a 100-fold greater potency. Unlike Eotaxin-1, Eotaxin-3 mRNA expression was not induced by either tumour necrosis factor (TNF)-alpha or interleukin (IL)-1 beta alone. Both IL-4 and IL-13 acted synergistically with TNF-alpha in superinducing Eotaxin-3 mRNA expression. Dexamethasone pre-treatment diminished induction of Eotaxin-3 mRNA expression. We conclude that modulation of Eotaxin-3 mRNA expression by Th(2) cytokines is different from that of Eotaxin-1 and Eotaxin-2, further supporting a distinct biological role for Eotaxin-3.     

The role of macrophage inflammatory protein-1 alpha/CCL3 in regulation of T cell-mediated immunity to Cryptococcus neoformans infection

Macrophage inflammatory protein-1alpha (MIP-1alpha/CCL3) is a CC chemokine required for optimal recruitment of leukocytes in response to cryptococcal Ags. MIP-1alpha is expressed in the lungs by day 6 post Cryptococcus neoformans infection and could play a role in the development of cell-mediated immunity. To address this possibility, wild-type (MIP-1alpha(+/+)) mice and MIP-1alpha knockout (MIP-1alpha(-/-)) mice were infected intratracheally with a highly virulent strain of C. neoformans (145A). MIP-1alpha message was detected in the lungs on days 3, 7, and 14 in MIP-1alpha(+/+) mice, but it was undetectable in MIP-1alpha(-/-) mice. On day 16, MIP-1alpha(-/-) mice had a 7-fold increase in C. neoformans burden in the lungs, but no decrease in pulmonary leukocyte recruitment. MIP-1alpha(+/+) and MIP-1alpha(-/-) mice had similar numbers of recruited lymphocytes and monocytes/macrophages. Notably, MIP-1alpha(-/-) mice had a significantly greater number of eosinophils. MIP-1alpha(-/-) mice had extremely high levels of serum IgE. This switch of immune response to a T(2) phenotype was associated with enhanced IL-4 and IL-13 expression in the lungs of MIP-1alpha(-/-) mice compared with MIP-1alpha (+/+) mice. Progression of pulmonary cryptococcosis in the presence of nonprotective T(2) immunity resulted in profound lung damage in MIP-1alpha(-/-) mice (eosinophilic crystal deposition, destruction of lung parenchyma, and pulmonary hemorrhage). Twelve-week survival was dramatically decreased in MIP-1alpha(-/-) mice. These studies, together with our previous studies, demonstrate that MIP-1alpha plays a role in both the afferent (T(1)/T(2) development) and efferent (T(1)-mediated leukocyte recruitment) phases of cell-mediated immunity to C. neoformans.     

Genomic organization of the CC chemokine mip-3alpha/CCL20/larc/exodus/SCYA20, showing gene structure, splice variants, and chromosome localization

We describe the genomic organization of a recently identified CC chemokine, MIP3alpha/CCL20 (HGMW-approved symbol SCYA20). The MIP-3alpha/CCL20 gene was cloned and sequenced, revealing a four exon, three intron structure, and was localized by FISH analysis to 2q35-q36. Two distinct cDNAs were identified, encoding two forms of MIP-3alpha/CCL20, Ala MIP-3alpha/CCL20 and Ser MIP-3alpha/CCL20, that differ by one amino acid at the predicted signal peptide cleavage site. Examination of the sequence around the boundary of intron 1 and exon 2 showed that use of alternative splice acceptor sites could give rise to Ala MIP-3alpha/CCL20 or Ser MIP-3alpha/CCL20. Both forms of MIP-3alpha/CCL20 were chemically synthesized and tested for biological activity. Both flu antigen plus IL-2-activated CD4(+) and CD8(+) T lymphoblasts and cord blood-derived dendritic cells responded to Ser and Ala MIP-3alpha/CCL20. T lymphocytes exposed only to IL-2 responded inconsistently, while no response was detected in naive T lymphocytes, monocytes, or neutrophils. The biological activity of Ser MIP-3alpha/CCL20 and Ala MIP-3alpha/CCL20 and the tissue-specific preference of different splice acceptor sites are not yet known.     

Impairment of mucosal immunity by total parenteral nutrition: requirement for IgA in murine nasotracheal anti-influenza immunity

Secretory IgA (SIgA) is the primary mucosal Ig and has been shown to mediate nasotracheal (NT) mucosal immunity in normal immune BALB/c mice. This finding has been challenged by a report of NT immunity without IgA in knockout mice, suggesting that IgA may not be necessary for the protection of mucosal surfaces. Although other protective mechanisms may become active in the congenital absence of SIgA, these mechanisms are not the primary means of protection in normal mice. In this paper we show that feeding chemically defined total parenteral nutrition (TPN) to genetically normal, immune ICR mice by the i.v. route results in loss of nasal anti-influenza immunity and a significant drop in influenza-specific SIgA in the upper respiratory tract compared with chow-fed mice (p < 0.005), while the serum influenza-specific IgG titer is unaffected. Loss of upper respiratory tract mucosal immunity is not related to serum Ab, because 10 of 13 TPN-fed mice shed virus into their nasal secretions despite adequate serum anti-influenza IgG titers. The number of IgG Ab-secreting cells in the nasal passages and spleens of TPN-fed mice was unaffected, while both the number and the percentage of splenic IgA-secreting cells were decreased relative to those in chow-fed animals. The loss of immunity is due to the route of nutrition, not the composition of the diet, because TPN solution fed orally via gastrostomy instead of i.v. maintains NT anti-influenza mucosal immunity. We hypothesize that delivery of nutrition via the gut triggers the release of gastrointestinal neuropeptides necessary for maintenance of the mucosal immune system.     

Ontogeny of testicular steroid dehydrogenase enzymes in pig (3 alpha/beta-, 20 alpha- and 20 beta-): evidence for two forms of 3 alpha/beta-hydroxysteroid dehydrogenase.

Three enzymatic activities (3 alpha/beta-hydroxysteroid dehydrogenase, 20 beta- and 20 alpha-hydroxysteroid dehydrogenases) were measured in testes of pigs as a function of age. Earlier studies reported a highly purified 20 beta-hydroxysteroid dehydrogenase from neonatal pig testes that also showed strong 3 alpha/beta-hydroxysteroid dehydrogenase activity [Ohno et al., J. Steroid Biochem. Molec. Biol. 38 (1991) 787-794]. We report here that neonatal pigs testis is rich in 3 alpha/beta- and 20 beta-hydroxysteroid dehydrogenase activities, both of which fall to low levels (measured as specific activity) at 60 days. Thereafter the activity of 3 alpha/beta-reduction rises to high levels whereas 20 beta-reduction remains low. Activity of 20 alpha-reduction is of intermediate level in the neonate, falls to a nadir at 60 days and rises to high levels in the mature animal. Western blots of cytosolic proteins show that the bifunctional enzyme (3 alpha/beta-plus 20 beta-hydroxysteroid dehydrogenase) is high in neonatal testes and falls to low levels at maturity. It is proposed that the neonatal testis possesses the bifunctional enzyme which is replaced by a second enzyme at maturity, that is a 3 alpha/beta-hydroxysteroid dehydrogenase without 20 beta-reductase activity. The possible functional significance of these changes is considered.     

Human receptor for measles virus (CD46) enhances nitric oxide production and restricts virus replication in mouse macrophages by modulating production of alpha/beta interferon

Complement regulatory protein CD46 is a human cell receptor for measles virus (MV). In this study, we investigated why mouse macrophages expressing human CD46 restricted MV replication and produced higher levels of nitric oxide (NO) in response to MV and gamma interferon (IFN-gamma). Treatment of MV-infected CD46-expressing mouse macrophages with antibodies against IFN-alpha/beta blocked NO production. Antibodies against IFN-alpha/beta also inhibited the augmenting effect of MV on IFN-gamma-induced NO production in CD46-expressing mouse macrophages. These antibodies did not affect NO production induced by IFN-gamma alone. These data suggest that MV enhances NO production in CD46-expressing mouse macrophages through action of IFN-alpha/beta. Mouse macrophages expressing a human CD46 mutant lacking the cytoplasmic domains were highly susceptible to MV. These cells produced much lower levels of NO and IFN-alpha/beta upon infection by MV, suggesting the CD46 cytoplasmic domains enhanced IFN-alpha/beta production. When mouse macrophages expressing tailless human CD46 were exposed to culture medium from MV-infected mouse macrophages expressing intact human CD46, viral protein synthesis and development of cytopathic effects were suppressed. Pretreating the added culture medium with antibodies against IFN-alpha/beta abrogated these antiviral effects. Taken together, these findings suggest that expression of human CD46 in mouse macrophages enhances production of IFN-alpha/beta in response to MV infection, and IFN-alpha/beta synergizes with IFN-gamma to enhance NO production and restrict viral protein synthesis and virus replication. This novel function of human CD46 in mouse macrophages requires the CD46 cytoplasmic domains.     

Tumor necrosis factor-alpha (TNF-alpha) induces integrin CD11b/CD18 (Mac-1) up-regulation and migration to the CC chemokine CCL3 (MIP-1alpha) on human neutrophils through defined signalling pathways

Strong evidence suggests that neutrophils may play an active role in acute and chronic inflammatory disorders, such as rheumatoid arthritis and atherosclerosis. Given the role of pro-inflammatory cytokine TNF-alpha in these inflammatory processes, we planned the present study to investigate the effect of short term incubation with TNF-alpha on neutrophil migration to CCL3, a chemokine produced in inflammatory sites and normally devoid of neutrophil chemotactic properties. We found that TNF-alpha primed neutrophils for migration to CCL3 via CCR5. TNF-alpha-induced migration was a consequence of the TNF-alpha-induced up-regulation of integrin CD11b/CD18 (Mac-1) on neutrophil surface. Furthermore, TNF-alpha activity was found to be strictly dependent on the activation of ERK 1/2 p44, cooperating with the intracellular pathways involving Src kinases, PI3K/Akt, p38 MAPK, well known as activated in response to classical chemoattractants (CXCL8) or priming agents (GM-CSF). On the contrary, the effect of TNF-alpha on neutrophil migration to CCL3 was not dependent on JNK 1/2. In conclusion, the present report shows that TNF-alpha unveils a previously unknown capacity of neutrophils to migrate to CCL3 through the intervention of Mac-1. TNF-alpha regulates Mac-1 up-regulation through signalling pathways, involving various kinases, but not JNK 1/2. Although highly speculative, ERK 1/2 p44 may represent a selective target for the pharmacologic manipulation of neutrophil-mediated adverse activities in TNF-alpha-mediated inflammatory states.     

20 beta-hydroxysteroid dehydrogenase of neonatal pig testis: 3 alpha/beta-hydroxysteroid dehydrogenase activities catalyzed by highly purified enzyme

Pig testicular 20 beta-hydroxysteroid dehydrogenase (20 beta-HSD) has also 3 alpha- and 3 beta-HSD (3 alpha/beta-HSD) activities. The purified 20 beta-HSD preparation from neonatal pig testes could catalyze the conversion of 5 alpha-dihydrotestosterone (5 alpha-DHT) in the presence of beta-NADPH to 5 alpha-androstane-3 alpha,17 beta-diol and 5 alpha-androstane-3 beta,17 beta-diol at the ratio of 4:3, and the specific 3 alpha/beta-HSD activity of 20 beta-HSD for 5 alpha-DHT was about 10 or 15 times larger than the 20 beta-HSD activities for 17 alpha-hydroxypregn-4-ene-3,20-dione (17 alpha-hydroxyprogesterone) or progesterone, respectively. The result indicates that the testicular 20 beta-HSD has high 3 alpha(axial, 3R)- and 3 beta(equatorial, 3S)-HSD activity. The testicular 20 beta-HSD could catalyze the reversible conversion of various 5 alpha- or 5 beta-dihydrosteroids which have a 3-carbonyl or 3-hydroxyl group with beta-NADP(H) as the preferred cofactor. The enzyme transferred the 4-proS hydrogen of NADPH to the 5 alpha-DHT for both 3 alpha- and 3 beta-hydroxylation and it was the same as the 20 beta-hydroxylation of 17 alpha-hydroxyprogesterone. Although the 3 alpha/beta-HSD activity has been known to be present in 3 alpha,20 beta-HSD of Streptomyces hydrogenans, the enzymological properties for 3 alpha/beta-HSD activity catalyzed by testicular 20 beta-HSD were different from the properties for 3 alpha/beta-HSD activity catalyzed by prokaryotic 3 alpha, 20 beta-HSD with respect to the specificity of the catalytic reaction and the cofactor requirement.