Usefulness of frequency analysis of cytotoxic T-lymphocytes precursors (CTL-p) for selection of HLA matched unrelated marrow donors.

A child with Sanfilippo syndrome and 5 potential unrelated marrow donors were typed serologically, tested in mixed lymphocyte reaction and typed by restriction fragment length polymorphism analysis in attempt to find a suitable donor. All donors were found to be identical with the recipient, however, these studies were not conclusive in identifying the best match donor. Therefore, recipient-donor pairs were examined by HLA-DR oligotyping. In addition we have studied the potential of cytotoxic T-lymphocytes precursors (CTL-p) analysis as a means of selection for matched unrelated donors. Low frequencies (1/10(5)) of pretransplant CTL-p correlated with oligotyping identity in all donor-recipient pairs evaluated. In one case oligotyping disclosed a previously unrecognized HLA-DRB1 disparity. This resulted in high frequencies of CTL-p (1/35,000). Based on this experience we can argue that CTL-p analysis may be used as an additional tool for selection of compatible unrelated marrow donors.

CD3-induced T-cell activation in the bone marrow of myeloma patients: major role of CD4+ cells.

A large expansion of activated T cells (CD3+CD25+) with the potential to act as anti-tumour effector cells is inducible in multiple myeloma (MM) patients by culturing bone marrow mononuclear cells (BMMCs) with the anti-CD3 monoclonal antibody (mAb) OKT3. The aim of this study was to provide a greater characterization of CD3-activated T cells. On day 6, most T cells coexpressed the CD11a, CD18, CD54, CD45R0 antigens and consisted of activated (CD25+) CD4+ and CD8+ cells in nearly equal proportions. Kinetics studies showed that CD4+CD25+ cells proliferated more rapidly and peaked earlier than CD8+CD25+ cells. When experiments were performed with purified subpopulations by removing CD4+ cells (resulting in CD8+ BMMCs) or by removing CD8+ cells (resulting in CD4+ BMMCs). T-cell activation and autologous plasma cell decrease were observed in CD4+ BMMCs only. Transwell cultures showed that CD4 help was necessary to make CD8+ BMMCs susceptible to CD3 stimulation. Relevant amounts of IL-2 were found in the supernatants of CD4+ BMMCs cultures, whereas no secretion of IL-4 was detected, indicating a Th1-like profile of CD3-activated CD4+ cells. These data indicate that CD4+ cells proliferate earlier and provide optimal help to induce the subsequent expansion of CD8+ cells after CD3 stimulation of MM BMMCs. Adequate stimulation of CD4+ cells is therefore essential in any strategy aiming to recover T-cell-mediated immunity in MM.

Dysregulated Fas and Bcl-2 expression leading to enhanced apoptosis in T cells of multiple myeloma patients.

We have previously reported the presence of activated (HLA-DR+) T cells in multiple myeloma (MM) patients. These cells produce high amounts of interleukin (IL)-2 and interferon (IFN)-gamma and generate a potent antiplasma cell activity after appropriate in vitro stimulation, but they are unable in vivo to hold in check the disease. Activated T cells are highly susceptible to apoptosis, a form of programmed cell death involved in the modulation of immune responses and regulated by molecules such as Fas (CD95) and bcl-2. The aim of this study was to determine the expression of Fas and bcl-2 antigens and the susceptibility to apoptosis in T cells of MM patients. Fas+ cells were significantly higher, whereas bcl-2+ cells were significantly lower in MM patients than in the controls. MM patients with the highest number of HLA-DR+ T cells showed the highest Fas and the lowest bcl-2 expression. Two-color cytofluorometric analysis confirmed in individual cells that HLA-DR+ T cells coexpressed Fas and lacked bcl-2. Susceptibility to apoptosis was then investigated to evaluate the consequence of dysregulated Fas and bcl-2 expression. The percentage of apoptotic cells after incubation in medium alone (spontaneous apoptosis) or in the presence of methylprednisolone (MP) or anti-Fas monoclonal antibody (triggered apoptosis) was significantly higher in MM and mainly restricted to HLA-DR+ T cells. Spontaneous apoptotosis was reverted by exogenous IL-2. In conclusion, MM T cells have a dysregulated expression of Fas and bcl-2 antigens that is associated with an enhanced susceptibility to apoptosis. These data may unravel a novel mechanism by which activated MM T cells are weakened in their ability to exert an effective antitumor activity in vivo.

Correlation between disease activity and T-cell CD3 zeta chain expression in a B-cell lymphoma.

The T-cell receptor (TCR)/CD3 complex is the surface molecule responsible for the highly specific response to foreign antigens, including tumour-associated antigens. and correlations have been observed with disease status. Recently, low amounts of CD3 gamma and CD3 zeta chains in the CD3/TCR complex have been reported in long-term tumour-bearing mice as a possible mechanism for the impaired T-cell responses. In this study we report a B-cell lymphoma in which T-cell CD3 zeta expression correlated with disease activity.

Generation of anti-tumour activity by OKT3-stimulation in multiple myeloma: in vitro inhibition of autologous haemopoiesis.

T cells in multiple myeloma (MM) patients are highly susceptible to activation with the anti-CD3 monoclonal antibody (mAb) OKT3. When short-term OKT3 stimulation is carried out on bone marrow mononuclear cells (BMMC), large numbers of CD3+ CD25+ HLA-DR+ cells are rapidly generated and autologous malignant plasma cells are killed. OKT3 may thus be exploited in autologous bone marrow transplantation (ABMT) to purge residual plasma cells and simultaneously activate T cells to induce graft-versus-leukemia-like (GVL-like) activity upon reinfusion. However, the possible impact of ex-vivo short-term OKT3 stimulation on haematological recovery is unknown. The aim of this work was to investigate the effect of OKT3 stimulation in vitro on autologous haemopoietic progenitor cells (HPC) of MM patients. Colony formation by granulocyte-macrophage progenitor cells (granulocyte-macrophage colony-forming units, CFU-GM) was highly suppressed, although supernatants of OKT3-activated T cells contained up to 2,500 pg/ml of granulocyte-macrophage colony-stimulating factor (GM-CSF). T cell depletion completely prevented this suppression. Neutralizing antibodies against TNF-alpha, TNF-beta and IFN-gamma (which are also produced by OKT3-activated MM T cells) did not prevent it, and Transwell cultures showed that cell-to-cell contact was the main mechanism involved. OKT3-activated T cells also suppressed erythroid burst-forming units (BFU-E) and CFU-GM generation from HPC responsible for long-term maintenance of in vitro myelopoiesis. When tested on normal allogeneic BM, MM supernatants of OKT3-stimulated BMMC partially suppressed the generation of day 7 CFU-GM, but had no effect on day 14 CFU-GM. These data indicate that short-term stimulation of BMMC with OKT3 can be used to generate anti-tumour effector T cells for autologous adoptive immunotherapy. It is not a feasable approach for ex-vivo purging and activation procedures in ABMT because of its potent inhibition of autologous haemopoiesis.

Co-stimulatory signal delivered by CD73 molecule to human CD45RAhiCD45ROlo (naive) CD8+ T lymphocytes.

CD73 is a molecule expressed by a subset of CD8+ human T lymphocytes and is involved in T cell activation. CD73 expression and function were analyzed in peripheral blood CD45RAhiCD45ROlo (naive) and CD45RAloCD45ROhi (memory) CD8+ cells. We found that CD73 was expressed by a majority of naive cells (74 +/- 12%), whereas fewer memory cells were CD73+ (29 +/- 10%). Moreover, CD73 was selectively expressed by the CD11b- subset of naive CD8+ cells, which were almost all CD73+. The same result was found on CD8+ cord blood lymphocytes, which prevalently display the naive phenotype. Naive CD8+ CD11b- cells were almost unresponsive to CD3 engagement, but this apparent anergy was completely overcome when CD3 and CD73 were simultaneously cross-linked by plastic-immobilized CD73 and CD3 mAb, showing that CD73 delivers an accessory signal that allows their activation via the CD3/TCR. This costimulatory signal was tenfold more potent than that induced by CD28 ligation. A phosphotyrosine analysis by Western blotting showed that cross-linking of CD73 induced the phosphorylation of two proteins with a molecular mass of approximately 28 and 100 kDa respectively, whereas ligation of CD3 induced phosphorylation of many substrates. When CD3 and CD73 were simultaneously triggered these substrates were hypophosphorylated. Because CD73 is linked to the cell surface by a GPI anchor, the transduction of this signal is probably mediated by a lateral interaction with transmembrane molecules. This hypothesis was assessed by cocapping, which showed that CD73 associates strongly with CD45RC, moderately with CD8, and weakly with CD3. These data suggest that CD73 signaling is coupled to both tyrosine kinase and phosphatase activities.

Rapid generation of antiplasma cell activity in the bone marrow of myeloma patients by CD3-activated T cells.

We have recently shown that peripheral blood T cells of multiple myeloma (MM) patients are very susceptible to stimulation of the T-cell receptor/CD3 complex with anti-CD3 monoclonal antibodies (MoAbs). CD3 stimulation is currently under clinical investigation as a nonspecific approach to boost antitumor effector mechanisms. The aim of this study was to determine whether the hyperreactivity of MM T cells to CD3 stimulation could be exploited to generate antitumor activity. Bone marrow mononuclear cells (BMMCs) from 65 MM patients were stimulated with the anti-CD3 MoAb OKT3 and the effect of this stimulation on autologous T cells and plasma cells was evaluated. The number of CD3+ CD25+ cells on day 6 was significantly higher in MM than the controls (30 normal individuals) (P = .001). Kinetic studies showed that 3H-thymidine incorporation peaked on day 3 and that the T-cell expansion peaked on days 5 and 6. In MM, T-cell activation markedly affected the survival of autologous plasma cells; their number in OKT3-treated cultures was significantly lower than in unstimulated cultures (P < .0001). T-cell activation and plasma cell decrease were not observed when T cells were removed from BMMC preparations. MM produced significantly higher levels of interferon-gamma (P = .005) and tumor necrosis factor-beta (P = .001), but lower levels of tumor necrosis factor-alpha (P < .001) than normal individuals. Interferon-gamma only was partially involved in CD3-induced plasma cell killing. Transwell cultures showed that the main mechanism by which CD3+ CD25+ cells affected plasma cells was direct cell-to-cell contact rather than cytokines. In conclusion, T cells in MM BMMCs possess distinct features in terms of susceptibility to CD3 stimulation and cytokine production compared with normal bone marrow T cells that can be exploited to generate antiplasma cell activity.

Constitutive production of tumor necrosis factor-alpha in hairy cell leukemia: possible role in the pathogenesis of the cytopenia(s) and effect of treatment with interferon-alpha.

PURPOSE: In view of the pleomorphic role cytokines play in human lymphoproliferative disorders, we investigated the possible involvement of tumor necrosis factor-alpha (TNF) in hairy cell leukemia (HCL). PATIENTS AND METHODS: The levels of TNF were measured in the serum of untreated patients, and in the culture supernatants of unstimulated and stimulated enriched hairy cells (HC). Furthermore, the presence of TNF mRNA transcripts in HC was analyzed. The possibility that HC could inhibit the in vitro growth of normal erythroid progenitors via the release of TNF was also investigated. Finally, in an attempt to correlate the circulating levels of TNF with the course of the disease, these were retested during and after treatment with interferon-alpha (IFN). RESULTS: Significantly increased levels of TNF were found in the sera of untreated HCL patients compared with normal control sera were seen from patients with other diseases (P less than .001), with values greater than 10 pg/mL in 21 of 42 samples tested. A significant decrease (P less than .01) of TNF levels was recorded following IFN-2a administration in 16 cases with detectable pretreatment serum levels of TNF. In two cases, an increase in TNF values was associated with persistence or progression of disease. The likelihood that the circulating levels of TNF were caused by the pathologic cells is supported by the evidence that purified HC may release TNF spontaneously. The values can be markedly increased following in vitro activation with the phorbol ester 12-0-tetradecanoylphorbol-13 acetate (PMA), with B-cell growth factor (BCGF), and, to a further extent, with the combination of PMA and BCGF. Furthermore, the constitutive mRNA for TNF was found in seven of eight HC samples analyzed. Although supernatants of enriched HC, were capable of reducing the growth of normal bone marrow erythroid progenitors by 50%, duplicate experiments using an anti-TNF antibody produced an almost complete disappearance of the inhibitory effect. CONCLUSION: The results of this study suggest that TNF plays an important role in the pathogenesis of the cytopenia(s) characteristically associated with HCL.

Detection of hyperreactive T cells in multiple myeloma by multivalent cross-linking of the CD3/TCR complex.

Cellular immunity was investigated in 43 patients with multiple myeloma (MM) by assessing 3HTdR uptake induced by monocyte-dependent [CD3 monoclonal antibodies (MoAbs), phytohemagglutinin (PHA)] and monocyte-independent (CD2 MoAbs, ionomycin + phorbolester) stimulations. The former were evaluated in peripheral blood mononuclear cells (PBMNC) and purified T cells; the latter were evaluated in purified T-cell preparations only. MM showed significantly lower PBMNC responses to PHA (P less than .001), soluble OKT3 (CD3) (P = .01), and immobilized OKT3 MoAbs (P = .01). On purification of T cells, MM responses were still defective to soluble T11(2) + T11(3) (CD2) MoAbs (P = .004), phorbol myristate acetate (PMA) plus ionomycin (P less than .001), but significantly higher to plastic-immobilized OKT3 (P = .004). In some MM, 3HTdR uptake, interleukin-2 (IL-2) receptor (CD25) expression, and IL-2 production were as high on stimulation with plastic-immobilized OKT3 as that observed in normal subjects under optimal conditions (ie, plastic-immobilized OKT3 plus accessory signals). CD3 hyperreactivity correlated with the number of CD8+ HLA-DR+ cells in MM T-cell preparations. MM patients with more than 10% CD8+ HLA-DR+ cells had significantly higher responses to immobilized OKT3 (P less than .001), but lower responses to T11(2) plus T11(3) (P = .01), and PMA plus ionomycin (P = .03) than patients with less than 10% CD8+ HLA-DR+ cells. Phenotyping of CD45RA (naive) and CD45R0 (memory) expressions in resting MM T cells showed a lower ratio of CD45RA to CD45R0 in both CD4 (P less than .05) and CD8 (P less than .001) subpopulations. These data indicate that (a) some MM T cells require significantly fewer accessory signals (if any) to express the IL-2 receptor fully, secrete IL-2, and proliferate on multivalent cross-linking of the CD3/TCR complex; and (b) this peculiar state of activation is associated with high HLA-DR expression in CD8+ lymphocytes.

Phenotypic and functional analysis of peripheral blood lymphocytes during interferon-alpha 2b therapy in multiple myeloma patients with low tumor mass.

BACKGROUND: IFN-alpha has recently been shown to prolong the remission phase in MM patients with low tumor mass. So far, it is not known whether IFN-alpha exerts its effect directly on the myeloma cells or is mediated by modulation of the host response. METHODS: The immune status of 12 multiple myeloma patients with low tumor mass (10 in remission phase, 2 with stage IA disease) was investigated by phenotypic and functional analyses before, after 3, and after 6 months of recombinant interferon-alpha 2b (IFN-alpha) therapy. RESULTS: Phenotyping of peripheral blood lymphocytes (PBL) revealed a significant decrease of HLA-DR+ (P = 0.01) and CD20+ (P = 0.04) cells after 6 months of therapy. Two-color phenotyping of purified T cell populations (PBT) showed a significant increase of CD4+ CD11b+ cells (P = 0.01) after 6 months of therapy. Functional analyses were carried out on PBL (NK cell-mediated cytotoxicity) and PBT (alloreactive cytotoxicity, CTL; IL2-induced cytotoxicity, LAK activity). NK and CTL activities were poorly influenced by IFN-alpha treatment, whereas LAK activity showed a significant increase (P = 0.007). Any significant association between these immunological changes and the disease status was questioned by the lack of differences between MM in relapse and MM with stable disease at the sixth month of IFN-alpha therapy. CONCLUSIONS: i) IFN-alpha in MM with low tumor mass may exert its therapeutic activity by directly acting on the tumor cells; ii) the parameters which have been used in this study are not appropriate to monitor the immunological effects (if any) of IFN-alpha therapy.

Dipyridamole in vitro suppresses the generation of T-cell cytotoxic functions: synergistic activity with cyclosporine.

The pharmacological activity of dipyridamole has been related to its ability to increase intracellular cAMP. Elevated cAMP concentrations can tone down T-cell effector functions. The aim of this study was to evaluate the dipyridamole effect in vitro on the generation of alloreactive cytotoxic and lymphokine-activated killer cells in normal T-cell subpopulations. Dipyridamole suppressed T-cell cytotoxic functions in a dose-dependent way. The kinetics of suppression showed that dipyridamole prevented the first step of cytotoxicity, i.e. activation of the lytic program following allogeneic or interleukin-2 stimulation. The ability of dipyridamole to interact with the immunosuppressive activity of cyclosporine was also investigated. By itself, cyclosporine suppressed the generation of alloreactive cytotoxicity, but not the generation of lymphokine-activated killer cells. A synergistic immunosuppressive activity between dipyridamole and cyclosporine was observed on the generation of alloreactive cytotoxic T lymphocytes.

Human T cell activation. Synergy between CD73 (ecto-5'-nucleotidase) and signals delivered through CD3 and CD2 molecules.

Interaction of the glycosyl phosphatidylinositol-linked differentiation Ag CD73 (ecto-5'-nucleotidase) with the CD73-specific mAb 1E9 generates agonistic signals that strongly synergize with T cell activation induced by CD3 and CD2 mAb. This synergy is observed only when 1E9 is immobilized on plastic and occurs in the absence of accessory cells or exogenous lymphokines. 1E9 induces a rapid (though transient) increase in [Ca2+]i in a minor proportion (20 to 30%) of unfractionated T lymphocytes (presumably CD73+ cells). However, this [Ca2+]i mobilization is not sufficient to fully activate CD73+ T cells, as shown by the requirement of additional signals such as CD3 or CD2 stimulation to initiate T cell proliferation. These signals cannot be substituted by the exogenous lymphokines, rIL-1, rIL-2, or rIL-4, or PMA (when T cells are rigorously depleted of monocytes). These data indicate that CD73 may behave as an accessory molecule regulating interactions between T cells and antigens or APC. A comparison was carried out with mAb 9.3 to the differentiation Ag CD28, another agonistic molecule with activating properties similar to CD73. Despite their lower percentage, the ability of CD73+ T cells to amplify the proliferation induced by CD3 or CD2 mAb was equivalent or even greater than that of CD28+ T cells. Once activated, CD73+ cells may recruit the remaining (CD73-) cells primed by CD3 or CD2 stimulation. Based on these data, we suggest that CD73+ T lymphocytes may be a specialized subset to amplify immune responses originated by the CD3 and CD2 activation pathways. Finally, the functional association between CD73 and integral membrane molecules like CD3 and CD2 suggests that GPI-anchored molecules may play a role in transmembrane signaling mediated by conventional second messenger systems.

Production of tumor necrosis factor-alpha by B-cell chronic lymphocytic leukemia cells: a possible regulatory role of TNF in the progression of the disease.

Tumor necrosis factor-alpha (TNF) is a cytokine that displays a pleomorphic array of effects on different cell populations. Evidence is presented that TNF may be constitutively produced by B-cell chronic lymphocytic leukemia (B-CLL) and hairy cell leukemia (HCL) cells and that it may play a relevant role in these diseases. These conclusions are based on the presence of circulating levels of TNF in the serum of 20 of the 24 patients tested (83.3%), while undetectable values were found in normal sera. The suggestion that the increased serum levels were due to the leukemic cell population is strengthened by the evidence that purified B-CLL and HCL cells may constitutively release variable degrees of TNF. These levels markedly increase after incubation with interferon gamma or phytohemagglutinin (PHA) plus phorbol myristate acetate (PMA). The cellular release of TNF by primary B-CLL cells was significantly (P less than .001) higher in B-CLL stage O-I patients compared with stage II-III patients. The demonstration that, in B-cell chronic lymphoproliferative disorders, the pathologic cells may release TNF was further confirmed by the presence of the mRNA for this cytokine in primary and/or in pre-activated cells. Recombinant TNF was capable of inducing a proliferative signal only in a minority of cases (4/24); in most cases it was ineffective, and, in a few, it reduced the degree of proliferation. Furthermore, in costimulatory experiments with interleukin-2 and PHA plus PMA, TNF was ineffective. On the other hand, when primary B-CLL cells were incubated in the presence of an anti-TNF antibody, in 8 of 12 independent experiments a 2- to 15-fold increase in thymidine uptake was documented. Taken together, these results suggest that TNF may play a regulatory role in the progression of the neoplastic clone in B-cell chronic lymphoproliferative disorders and may be implicated in some of the side effects associated with these diseases.

Defective interleukin-2 induction of lymphokine-activated killer (LAK) activity in peripheral blood T lymphocytes of patients with monoclonal gammopathies.

The recombinant interleukin-2 (rIL-2) generation of lymphokine-activated killer (LAK) cells was investigated in peripheral blood T lymphocytes (PBT) of 16 patients with monoclonal gammopathy of undetermined significance (MGUS) and 32 patients with multiple myeloma (MM). LAK activity was significantly decreased in MM, but not in MGUS patients, and was partially recovered in MM in the remission phase. This finding was unexpected, because CD8+ CD11b+ cells, which contain LAK precursors, are significantly increased in MM. LAK activity was investigated in purified CD8+ CD11b+ lymphocytes to discriminate between an intrinsic defect or a defective regulation by other T cell subsets. These cells were intrinsically unable to generate LAK activity fully following rIL-2 stimulation. MM showed the more pronounced LAK deficiency, while MGUS patients showed intermediate values. Phenotyping revealed significantly increased proportions of Leu7+ and HLA-DR+ cells in MM patients. These data reveal another dysregulation of T cell effector functions in patients with monoclonal gammopathies and offer further evidence of the impairment of their cell-mediated immunity.

Human liver microsomal steroid metabolism: identification of the major microsomal steroid hormone 6 beta-hydroxylase cytochrome P-450 enzyme.

Cytochrome P-450-dependent steroid hormone metabolism was studied in isolated human liver microsomal fractions. 6 beta hydroxylation was shown to be the major route of NADPH-dependent oxidative metabolism (greater than or equal to 75% of total hydroxylated metabolites) with each of three steroid substrates, testosterone, androstenedione, and progesterone. With testosterone, 2 beta and 15 beta hydroxylation also occurred, proceeding at approximately 10% and 3-4% the rate of microsomal 6 beta hydroxylation, respectively, in each of the liver samples examined. Rates for the three steroid 6 beta-hydroxylase activities were highly correlated with each other (r = 0.95-0.97 for 25 individual microsomal preparations), suggesting that a single human liver P-450 enzyme is the principal microsomal 6 beta-hydroxylase catalyst with all three steroid substrates. Steroid 6 beta-hydroxylase rates correlated well with the specific content of human P-450NF (r = 0.69-0.83) and with its associated nifedipine oxidase activity (r = 0.80), but not with the rates for debrisoquine 4-hydroxylase, phenacetin O-deethylase, or S-mephenytoin 4-hydroxylase activities or the specific contents of their respective associated P-450 forms in these same liver microsomes (r less than 0.2). These correlative observations were supported by the selective inhibition of human liver microsomal 6 beta hydroxylation by antibody raised to either human P-450NF or a rat homolog, P-450 PB-2a. Anti-P-450NF also inhibited human microsomal testosterone 2 beta and 15 beta hydroxylation in parallel to the 6 beta-hydroxylation reaction. This antibody also inhibited rat P-450 2a-dependent steroid hormone 6 beta hydroxylation in uninduced adult male rat liver microsomes but not the steroid 2 alpha, 16 alpha, or 7 alpha hydroxylation reactions catalyzed by other rat P-450 forms. Finally, steroid 6 beta hydroxylation catalyzed by either human or rat liver microsomes was selectively inhibited by NADPH-dependent complexation of the macrolide antibiotic triacetyloleandomycin, a reaction that is characteristic of members of the P-450NF gene subfamily (P-450 IIIA subfamily). These observations establish that P-450NF or a closely related enzyme is the major catalyst of steroid hormone 6 beta hydroxylation in human liver microsomes, and furthermore suggest that steroid 6 beta hydroxylation may provide a useful, noninvasive monitor for the monooxygenase activity of this hepatic P-450 form.

Monoclonal antibodies inhibitory to rat hepatic cytochromes P-450: P-450 form specificities and use as probes for cytochrome P-450-dependent steroid hydroxylations.

Cytochrome P-450 (P-450) form specificities were established for a total of nine monoclonal antibodies (MAbs) raised to four distinct rat hepatic P-450 enzymes (P-450s 2c, PB-2a, PB-4, and BNF-B), using a combination of enzyme-linked immunosorbent analysis, dot immunoblotting, Western blotting, Ouchterlony immunodiffusion, and immunoinhibition analyses. Four of the MAbs were fully (greater than or equal to 85%) inhibitory toward the corresponding immunoreactive P-450s when assayed in purified, reconstituted enzyme systems, while two of the MAbs were partially inhibitory, with a maximum of 50 or 80% inhibition achieved in the presence of saturating MAb. Inhibitory MAbs reactive with P-450s 2c, 3, and PB-4, respectively, were used to demonstrate that the formation of multiple hydroxytestosterone metabolites by each of the respective purified P-450 enzymes is reflective of their inherent catalytic specificities and not due to the presence of immunochemical distinguishable P-450 enzyme contaminants. P-450 form-specific contributions to rat hepatic microsomal steroid hormone hydroxylase activities were then assessed using the inhibitory MAbs as probes. MAb-reactive P-450 2c was shown to be the major (greater than or equal to 85%) catalyst of microsomal testosterone and androstenedione 16 alpha-hydroxylation in both untreated and beta-naphthoflavone-induced rats. However, this P-450 form catalyzed only approximately 30% of hepatic microsomal steroid 16 alpha-hydroxylase activity in phenobarbital-induced adult males, and less than or equal to 10% of steroid 16 alpha-hydroxylase activity in (phenobarbital-induced immature males or adult females, where the balance of 16 alpha-hydroxylase activity is catalyzed by MAb-reactive P-450 PB-4. Although MAb-reactive P-450 PB-4 catalyzed the majority (greater than or equal to 90%) of microsomal androstenedione 16 beta-hydroxylation in phenobarbital-induced rats, this P-450 enzyme did not contribute to the low level 16 beta-hydroxylase activity of uninduced liver samples. Finally, MAb-reactive P-450 3 catalyzed at least 85% of microsomal androstenedione 7 alpha-hydroxylation, independent of the age, sex, or induction status of the animals used as source of liver microsomes. These findings demonstrate the usefulness of MAbs as probes for the contributions of individual P-450 enzymes to the metabolism of steroid hormones susceptible to hydroxylation at multiple sites.

Preparation and characterization of monoclonal antibodies to pregnenolone 16-alpha-carbonitrile inducible rat liver cytochrome P-450.

Hybridomas were prepared from mouse myeloma cells and spleen cells derived from BALB/c female mice immunized with purified rat hepatic pregnenolone 16-alpha-carbonitrile (PCN) induced cytochrome P-450 2a/PCN-E. The monoclonal antibodies (MAbs) thus obtained were screened for binding to the purified P-450 2a/PCN-E by radioimmunoassay. Eleven independent hybrid clones produced MAbs, each of which was of a single mouse immunoglobulin subclass of the IgG1, IgG2a or IgG2b type. Each of the MAbs produced by the eleven individual hybrid clones bound strongly to P-450 2a/PCN-E as assessed by radioimmunoassay and immunoprecipitation of P-450 2a/PCN-E in Ouchterlony double-immunodiffusion plates. Of the eleven MAbs, three also bound strongly to the phenobarbital-inducible rat liver cytochrome P-450 PB-4. Thus, two classes of MAbs were obtained, one class specific for P-450 2a/PCN-E and a second class that bound to both PCN- and phenobarbital-inducible P-450 forms. The reactivities of one MAb from each class toward eight highly purified rat hepatic cytochromes P-450 were examined using solid phase enzyme-linked immunosorbent analyses. The MAb designated C2 was found to be specific for P-450 2a/PCN-E and did not cross-react with seven other P-450 forms. This MAb was shown to be an effective probe for monitoring, by Western blotting, the induction of microsomal P-450 2a/PCN-E by PCN and phenobarbital. The MAb designated C1 reacted both with P-450 2a/PCN-E and with the two major phenobarbital-inducible P-450 forms, PB-4 and PB-5. None of the MAbs was inhibitory towards P-450 2a/PCN-E-dependent aryl hydrocarbon hydroxylase, benzphetamine N-demethylase, ethoxycoumarin O-deethylase or ethymorphine N-demethylase activity, indicating that the epitopes recognized by these MAbs are not directly associated with catalytic activity. The strong reactivities of three of the MAbs with both P-450 2a/PCN-E and P-450s PB-4 and PB-5 indicate that these two structurally quite different cytochrome P-450 families share at least one common epitope. These new MAbs are additions to our library of MAbs to different cytochromes P-450 and should help further our understanding of the relationship of cytochrome P-450 phenotype and multiplicity to inter-individual differences in drug and carcinogen metabolism and sensitivity.