Abnormal thymic maturation and lymphoproliferation in MRL-Fas lpr/lpr mice can be partially reversed by synthetic oligonucleotides: implications for systemic lupus erythematosus and autoimmune lymphoproliferative syndrome.

MRL-Fas lpr/lpr mice represent an excellent animal model for studying non-malignant lymphoproliferation, regeneration and systemic autoimmunity. Retro-transposon insertion into the second intron of the pro-apoptotic Fas gene appears to be responsible for both lymphoproliferation and autoimmunity, while other genes are more likely to contribute to the regenerative healing characteristic of this mouse strain. Previous studies have shown that neonatal thymectomy can halt the development of abnormal lymphoproliferation. Whereas at four weeks of age primary and secondary lymphoid organs appear to be grossly intact, vigorous lymphoproliferation and autoantibody production subsequently ensues. This is first noticeable at six weeks of age, at which time lymph nodes, spleens and thymuses, but not the bone marrow, become infiltrated with abnormal B220+CD3+CD4-CD8- T cells. Around the same time, thymuses show a significant drop in CD4+CD8+double-positive T cells generating an abnormal ratio between double-positive and single-positive thymocytes. The objective of current study was to evaluate the effect of synthetic oligonucleotides-toll-like receptor antagonists on early lymphoid development in this strain of mice. Herein, we demonstrate the ability of synthetic oligonucleotides made with the nuclease-resistant phosphorothioate backbone to partially reverse abnormal lymphoproliferation and thymic involution in pre-diseased MRL-Fas lpr/lpr mice when administered intraperitoneally starting from week four of age. This curative effect of oligonucleotides was primary sequence/secondary oligonucleotide structure-independent, suggesting an effect through the toll-like receptor 7. A similar approach may potentially benefit patients with autoimmune lymphoproliferative syndrome who, like MRL-Fas lpr/lpr mice, carry a mutation in the Fas gene.

CpG stimulation of primary mouse B cells is blocked by inhibitory oligodeoxyribonucleotides at a site proximal to NF-kappaB activation.

Bacterial DNA and CpG-oligodeoxyribonucleotides (ODN) are powerful B cell activators, inducing apoptosis protection, cell cycle entry, proliferation, costimulatory molecule expression, immunoglobulin (Ig) and interleukin-6 (IL-6) secretion. However, proximal events in B cell activation by ODN are only partially characterized, including the translocation of NF-kappaB to the nucleus. In this paper, we provide evidence that CpG-ODN-induced cell cycle entry and apoptosis protection are blocked by SN50 or gliotoxin and thus require NF-kappaB activation. NF-kappaB activation occurred within 30 minutes of stimulation of murine B cells with a phosphorothioate (S) CpG-ODN and persisted for up to 40 hours, with p50, p65, and c-Rel as the major components. Similar to other NF-kappaB inducers, CpG-ODN caused an early IkappaBalpha and IkappaBbeta degradation plus cleavage of the p50 precursor and subsequent NF-kappaB nuclear translocation. A group of closely related S-ODN, which specifically blocked CpG-induced B cell activation at submicromolar concentrations, also prevented NF-kappaB DNA binding and transcriptional activation. These inhibitory S-ODN differed from stimulatory S-ODN by having 2-3 G substitutions in the central motif. As inhibitory S-ODN did not directly interfere with the NF-kappaB DNA binding but prevented CpG-induced NF-kappaB nuclear translocation of p50, p65, and c-Rel and blocked p105, IkappaBalpha, and IkappaBbeta degradation, we concluded that their putative target must lie upstream of inhibitory kinase (IKK) activation.

Difference in apoptosis induction between surface IgD and IgM.

In the classic 'two-signal' model for B cell activation, signal 1 through the antigen receptor plus signal 2 through lymphokine receptors and CD40 leads to proliferation, but signal 1 alone leads to tolerance or anergy. In a protocol designed to deliver signal 1 in vitro with anti-delta without signal 2, purified small dense B cells from untreated mice exposed to any of three monoclonal anti-delta antibodies or to polyclonal anti-delta in vitro showed modest S phase entry at 50 microg/ml. In contrast, at low doses (0.1-0.5 microg/ml) of anti-delta, there was no cell cycle entry at 64 h, but apoptosis was accelerated at 16 h. Polyclonal anti-mu and three monoclonal anti-mus did not show this early apoptosis induction. Anti-CD40 and IL-4 inhibited apoptosis in B cells treated with 0.5 microg/ml anti-delta and increased S phase entry at 10 microg/ml anti-delta. Low-dose anti-delta (but not anti-mu) induced increased B7-2 and class II MHC expression on a subset of B cells, many of which were in apoptosis. Larger transient increases in c-Myc and Egr-1 expression were seen with low-dose anti-delta than with anti-mu, followed by an abrupt fall below baseline, a sequence previously linked to apoptosis. There was no change in Bcl-2, Bcl-x(L) or Bax. These data suggest a functional difference between delta and mu cross-linking on resting spleen B cells. A BCR stimulus sufficient for early activation events, but insufficient for full G1 entry, may lead to apoptosis.

CpG DNA rescues B cells from apoptosis by activating NFkappaB and preventing mitochondrial membrane potential disruption via a chloroquine-sensitive pathway.

Isolated murine splenic B cells gradually undergo spontaneous apoptosis while WEHI-231 B lymphoma cells undergo activation-induced apoptosis. Unmethylated CpG dinucleotides in a particular sequence context (CpG motif) in bacterial DNA or in synthetic oligodeoxynucleotides (CpG DNA) rescue both splenic B cells and WEHI-231 cells from apoptosis, an effect which could potentially contribute to autoimmune disease. Chloroquine has been used as an effective therapeutic agent for some autoimmune diseases, although the mechanism of action is not clearly understood. Low concentrations of chloroquine (<5 microM) selectively abolished CpG DNA-mediated protection against spontaneous apoptosis of splenic B cells and against anti-IgM-induced apoptosis of WEHI-231 cells without affecting anti-apoptotic activities of anti-CD40 or lipopolsaccharide. CpG DNA effectively prevented mitochondrial membrane potential disruption through a chloroquine-sensitive pathway in splenic B cells. Apoptosis protection by CpG DNA was also associated with increased expression of several proto-oncogenes and oncoproteins directly and/or indirectly through a rapid and sustained activation of NFkappaB in splenic B cells and WEHI-231 cells. These effects were also suppressed by chloroquine. Our results suggest that despite the difference in maturation phenotype of splenic B cells and WEHI-231 cells, CpG DNA rescues both from apoptosis by similar pathway, which is blocked at an early step by chloroquine.

CpG oligodeoxyribonucleotides rescue mature spleen B cells from spontaneous apoptosis and promote cell cycle entry.

Isolated murine splenic B cells undergo spontaneous apoptosis. Motifs containing unmethylated CpG dinucleotides in bacterial DNA or in synthetic oligodeoxynucleotides (ODN) are known to activate murine B cells. Now we show that ODN that induce spleen B cell cycle entry also inhibit spontaneous apoptosis in a sequence-specific fashion. Reversal of the CG to GC abolished activity. Methylation of the central cytosine decreased activity. When CpG is preceded by a cytosine or followed by a guanine, activity was abolished. Other substitutions at the same positions had no effect. Dose-response curves for apoptosis protection and G1 entry suggested that a uniform population of ODN recognition sites controlled downstream ODN effects. A CpG ODN with a nuclease-resistant phosphorothioate backbone (S-ODN) was also active, and increased the levels of c-myc, egr-1, c-jun, bclXL, and bax mRNA and c-Myc, c-Jun, Bax, and BclXL protein in spleen B cells. Levels of c-myb, myn, c-Ki-ras, and bcl2 mRNA remained unchanged. When protein synthesis was inhibited, at 16 h ODN-induced cell cycle entry was abolished and apoptosis protection was partially preserved. Under these conditions, c-Myc was still present, but c-Jun and BclXL were not detected. Our results suggest that CpG containing ODN motifs provide signals for both survival and cell cycle entry. Single base changes determine whether this signal proceeds through a rate-limiting step governing at least two steps in apoptosis (plasma membrane transition, DNA cleavage) and two phases of the cell cycle (G1 and S phase entry). This biologic action is associated with increased c-Myc, c-Jun, and BclXL expression.

Direct evidence that iron deprivation induces apoptosis in murine lymphoma 38C13.

We found that the mouse B cell lymphoma 38C13 underwent apoptosis in vitro when deprived of iron by three independent methods: (1) exposure to a synergistic pair of rat IgG monoclonal antibodies against the mouse transferrin receptor; (2) exposure to the iron chelator deferoxamine (DFO), and (3) exposure to a defined culture medium without any added iron (iron-poor medium). When each antibody was present at a concentration of 5 micrograms/ml, the number of living cells declined to approximately 25% after a 24-hour incubation. After 48 h, there were no surviving cells. When DFO was present at a concentration of 10 microM, the effects were similar, but delayed by 24 h. when iron-poor medium was used, the effects and kinetics were similar to those seen with antibody treatment. For each method of iron deprivation, the reduction in cell viability correlated with the development of apoptosis, as assessed by DNA fragmentation analysis and propidium iodide staining. Electron microscopy studies provided additional confirmation of apoptotic cell death. The addition of 500 microM ferric citrate completely prevented apoptosis for each of the three methods of iron deprivation. These studies provide new and compelling evidence to support the view that iron deprivation can specifically induce apoptosis and serve to strengthen the rationale for further studies of iron deprivation as a form of cancer treatment.

Fc receptor off-signal in the B cell involves apoptosis.

By linking surface Ig to the FcR Fc gamma RII on the mouse B lymphocyte surface, whole anti-Ig has been shown to block cell cycle entry and subsequent Ab production, a phenomenon called the "Fc receptor off-signal." IL-4 or blocking Ab to Fc gamma RII, present with whole anti-Ig, restores cell cycle progress to the levels observed with F(ab')2 anti-Ig. The current study demonstrates that under "off-signal" conditions with whole anti-Ig, the early entry of B cells into apoptosis was accelerated relative to medium alone or equimolar F(ab')2 anti-Ig. All reagents tested which opposed the whole-anti-Ig-induced blockade of B cell cycle entry also protected B cells from apoptosis (IL-4, PMA, dextran sulfate, and the monoclonal anti-Fc gamma RII 2.4G2). This protective effect was most evident at 4 to 12 h, waning at later times. Low dose cycloheximide partially protected B cells from whole anti-Ig-induced apoptosis, but acted as a survival factor, failing to advance B cells from G0 phase or stimulate thymidine incorporation. Additive early apoptosis-associated membrane changes were transiently seen when whole anti-Ig was combined with other apoptosis-accelerating agents (trifluoperazine, staurosporine, dexamethasone, ionomycin, high-dose cycloheximide), but hypodiploid nuclei did not show this effect. B cells from bcl-2 transgenic mice showed less apoptosis when cultured with whole anti-Ig, or with any of the other agents tested. At 4 h the bcl-2-associated reduction in hypodiploid nuclei was greater than the reduction in membrane unpacking, but by 16 h this difference was much less. These results suggest that acceleration of apoptosis contributes to the inhibition of proliferation induced by whole anti-Ig.

Membrane unpacking and the rapid disposal of apoptotic cells.

There is evidence that macrophages can recognize phosphatidylserine (PS) on the surface of apoptotic thymocytes, where PS exposure relates to looser packing ('unpacking') of the polar headgroups of the outer lamina, detectable by lipophilic dyes (Fadok, V.A., Voelker, D.R., Campbell, P.A., Cohen, J.J., Bratton, D.L. and Henson, P.M. (1992) J. Immunol. 148, 2207). We have shown that membrane unpacking also occurs in B cells, where this event actually precedes DNA cleavage (Mower, D.A. Jr., Peckham, D.W., Illera, V.A., Fishbaugh, J.K., Stunz, L.L. and Ashman, R.F. (1994) J. Immunol. 152, 4832). This paper demonstrates that the time interval between membrane unpacking (detected as merocyanine 540 binding) and DNA cleavage (detected by flow cytometry of propidium iodide stained nuclei) also occurs in both T cells and thymocytes. The tight coupling of these two apparently distinct events is emphasized by their co-regulation by a variety of agents which accelerate or inhibit apoptosis. One hypothesis to explain the very low numbers of free apoptotic cells seen in vivo is that macrophages can recognize cells with unpacked membranes and destroy them before they cleave their DNA. In support of this hypothesis, we demonstrated that parenteral cycloheximide triggers a wave of apoptosis in the spleen detected by merocyanine 540 as well as by hypodiploid nuclei. Significantly, both parameters returned from peak values at 2 h virtually to normal by 4 h, testifying to the existence of a rapid disposal mechanism in vivo for cells with unpacked membranes as well as hypodiploid nuclei.

Decreased membrane phospholipid packing and decreased cell size precede DNA cleavage in mature mouse B cell apoptosis.

Mature resting mouse spleen B cells progress stochastically into apoptosis at a uniform rate over the first 16 h in vitro in 3 stages. In stage 1, early apoptotic B cells decreased the normal phospholipid packing of their plasma membranes, detected as increased binding of the lipophilic dye merocyanine 540, and also decreased in volume, detected as decreased forward scatter. In stage 2 there was abrupt internucleosomal cleavage of DNA, quantitated as hypodiploid nuclei by flow cytometry. Some stage 2 cells entered stage 3, where the plasma membrane became permeable to propidium iodide. B cells in later stages of this sequence retained the characteristics of earlier stages, whereas nonapoptotic B cells remained in their original state. Cycloheximide increased the progression of B cells through these three stages, whereas dextran sulfate inhibited stage 1 more effectively than stages 2 or 3. Increased orthogonal scatter also occurred late in some of the cells that had passed through stage 1, but did not correlate well with propidium iodide permeability. Fresh small dense spleen B cells contained 5% to 7% stage 1 cells but only about 1% stage 2 cells. Macrophages have been reported to destroy preferentially apoptotic thymocytes by recognizing plasma membrane alterations deriving from loose packing of phospholipid head groups. The recognition of stage 1 rather than stage 2 B cells by macrophages may help to keep the proportion of apoptotic cells low in vivo.

Analysis of high and low responses to Staphylococcus aureus and interleukin 2 in human B lymphocytes.

Fixed protein A-bearing staphylococci (SAC) stimulate human B cells via surface Ig, whereas IL-2 has been reported to provide a sufficient second signal for proliferation and differentiation. Using an ELISPOT assay to count cells secreting IgM, IgA, and IgG and flow cytometry with acridine orange to assess cell cycle progress, we have found that the purified B lymphocytes of a substantial minority (5/13) of healthy volunteers with normal serum Ig levels failed to differentiate to Ig secreting cells (ISC) in response to SAC + IL-2 (IgM, IgA, or IgG secreting cells, < 5% of input B cells). High-responders generally formed 10-35% ISC. The proportions of B cells expressing IgG, IgA, IgM, or IgD were not different in the two groups. By average linkage cluster analysis, SAC/IL-2 high- and low-responders were shown to fall into two separate populations with respect to ISC. High- and low-responders tended to remain in the same group with repeated testing over several months, although some convergence was seen. The low-responders also showed significantly less advancement to late G1 and S phase than the high-responders, in the presence of SAC +/- IL-2. Induction of IL-2 receptors on B cells by SAC + IL-2 was much greater in high-responders than in low-responders, as shown by flow cytometry with phycoerythrin-conjugated IL-2. However, SAC + IL-2 induced transferrin receptors normally in low-responders, showing that some early activation steps occur in these cells. Low-responder B cells often improved their responses in the presence of macrophages and T cell supernatants. Finally, bypassing the surface Ig pathway using anti-CD3-activated T cells to stimulate B cells produced normal differentiation in low-responder B cells. Thus a subset of clinically normal individuals possesses B cells which fail to express IL-2 receptors, proliferate, and differentiate normally in vitro in response to SAC + IL-2 yet can respond well to alternative activation pathways via T cells, monocytes, and their products.

Regulation of apoptosis in vitro in mature murine spleen T cells.

Previously it has been shown that thymocytes undergo apoptosis, a form of programmed cell death, in response to glucocorticoids. This classic form of apoptosis is prevented by inhibition of protein synthesis. The current paper demonstrates that mature T cells also undergo apoptosis, but that the regulation of apoptosis in spleen T cells differs from that of thymocytes. Mature mouse spleen T cells were shown to die by apoptosis, not necrosis, when cultured without an added stimulus. Assays for apoptosis included internucleosomal DNA cleavage by gel electrophoresis, percent fragmentation of DNA by the diphenylamine method, and percent of cells with hypodiploid DNA by flow cytometry. The percent of apoptotic cells was 2% in fresh spleen T cells, and increased at least until 16 h, when 21% were apoptotic. Dexamethasone caused apoptosis in both thymus and spleen T cells, but only thymocytes showed a requirement for protein synthesis in dexamethasone-induced death. Cycloheximide increased apoptosis in spleen T cells, indicating that apoptosis was controlled by newly synthesized protective proteins. Spontaneous apoptosis was decreased in spleen T cells by protein kinase C activation, and was increased by H7 and staurosporine, which inhibits protein kinases, in contrast with the behavior of thymocytes. The protein kinase A/G inhibitor HA1004 also decreased spleen T cell apoptosis. The contrasting effects of cycloheximide on thymocytes and spleen T cells occurred over the same concentration range, and the same was true for PMA. The dexamethasone dose-response curves were similar, except that a greater proportion of spleen T cells were dexamethasone-resistant. These data support the hypothesis that the apoptosis program in T cells undergoes a transition during their maturation, such that apoptosis in mature T cells is regulated more like that of mature B cells than that of thymocytes.

Apoptosis in splenic B lymphocytes. Regulation by protein kinase C and IL-4.

Small dense splenic B lymphocytes from adult specific pathogen-free mice were shown to undergo apoptosis in vitro as indicated by internucleosomal DNA fragmentation, hypodiploid DNA content of isolated nuclei, and morphologic features by electron microscopy. Unstimulated cultures showed spontaneous apoptosis increasing gradually and monotonically from < 2 to 32% of B cells by 16 h. The rate of accumulation of apoptotic cells was reduced by the addition of IL-4 or PMA, but not by the inactive phorbol ester, 4 alpha PDD. In contrast, inhibitors of protein kinase C (H7 and staurosporine) increased the percentage of cells undergoing apoptosis to > 70% by 12 h; HA 1004, genistein, and herbimycin A all had no effect on apoptosis. Thus, protein kinase C activity regulates apoptosis, but there is no evidence that protein kinases A and G and tyrosine kinases are involved. Cycloheximide increased apoptosis, indicating that apoptosis may be restrained in B cells by the presence of one or more labile protective proteins. The percentage of apoptotic cells measured by flow cytometry and the percentage of fragmented DNA measured by the diphenylamine method were nearly equal, regardless of the method of apoptotic regulation. Together with the absence of nuclei with flow cytometric properties intermediate between normal and apoptotic, these results suggest that in individual B cells apoptosis progresses rapidly to completion. These data suggest a fundamental change in our concept of the life-style of the "resting" B cell: instead of a dormant cell remaining unchanged until it receives activation signals, the mature spleen B cell appears programmed to die by apoptosis unless rescued by specific agents, such protein kinase C activators or IL-4.

Genetic and immunologic analysis of a family containing five patients with common-variable immune deficiency or selective IgA deficiency.

A family with 13 members included 2 subjects with selective IgA deficiency (IgA-D) and 3 subjects with common-variable immune deficiency (CVID), diseases which usually occur sporadically. Reciprocal combinations of B and T cells in vitro between one normal and two immune-deficient family members and normal subjects revealed that defective Ig synthesis was determined by the B cells, while the patient T cells functioned normally. Normal T helper and suppressor function was demonstrated even in one patient with CVID who developed a T-cell lymphoproliferative disorder associated with elevated IgM; this patient's B cells made only IgM in vitro. Immune deficiencies were inherited in this family in a pattern consistent with an autosomal dominant trait with incomplete penetrance. All the immune-deficient patients in this family possessed at least one copy of an MHC haplotype previously shown to be abnormally frequent in IgA-D and CVID: HLA-DQB1*0201, HLA-DR3, C4B-Sf, C4A-deleted, G11-15, Bf-0.4, C2-a, HSP70-7.5, TNF alpha-5, HLA-B8, and HLA-A1. The patient who developed the lymphoproliferative disorder was homozygous for this haplotype. Four immunologically normal members, one of whom was 80 years old, also possessed this MHC haplotype, indicating that its presence is not sufficient for disease expression. A small segment of another MHC haplotype associated with Ig deficiency in the population also occurred in this family, but it was not associated with immune deficiency.(ABSTRACT TRUNCATED AT 250 WORDS)

Actin polymerization in murine B lymphocytes is stimulated by cytochalasin D but not by anti-immunoglobulin.

One might predict that cytochalasin D, which slows polymerization of actin in solution and which inhibits actin-containing microfilament function in live B lymphocytes, would also prevent actin polymerization in these cells. However, we have used the NBD-Phallacidin flow cytometric assay for F-actin and the DNase I inhibition assay for G-actin to demonstrate that cytochalasin D (at 20 micrograms/ml and higher) stimulates actin polymerization in murine B lymphocytes within the first 30 sec of exposure. A similar response was seen in human neutrophils. Actin polymerization induced in neutrophils by chemotactic peptides has been linked to activation of the polyphosphoinositide-calcium increase-protein kinase C signal transduction pathway. As B lymphocytes also transduce signals using this pathway, we investigated whether cytochalasin D induced actin polymerization by activating this pathway. Cytochalasin D and ionomycin both stimulated a rapid increase in internal calcium (by 1 min) in the B cell which was inhibitable by EGTA, implicating calcium influx. Ionomycin also induced actin polymerization, detectable later, by 10 min. EGTA blocked the ionomycin-induced actin polymerization, but not that induced by cytochalasin D. Cytochalasin D-induced actin polymerization was not associated with detectable hydrolysis of polyphosphoinositides, nor was it inhibited by H7 (a protein kinase C inhibitor) or by HA1004 (an inhibitor of cyclic nucleotide-dependent kinases). Furthermore, anti-immunoglobulin antibodies, which stimulate B lymphocytes through the polyphosphoinositide hydrolysis-calcium increase-protein kinase C pathway, failed to induce actin polymerization in these cells. These antibodies did, however, stimulate the cells to perform activities that involve actin-containing microfilaments. Other primary activators of B lymphocytes (dextran sulfate, PMA, and LPS) and a panel of lymphokines previously shown to enhance B lymphocyte activation (IL-1, IL-2, IL-4, IL-5) were also screened in the F-actin assay and no evidence for actin polymerization was found. We conclude that the actin polymerization response to cytochalasin D in the B cell does not involve the polyphosphoinositide hydrolysis-calcium increase-protein kinase C pathway, nor does it depend on cyclic nucleotide-dependent kinases. Furthermore, our studies failed to provide any evidence that early actin polymerization occurs in murine B lymphocyte activation.

Two mechanisms of transferrin receptor induction by anti-Ig in B cells.

By cross-linking surface Ig to the Fc gamma R, whole (IgG)-rabbit anti-Ig antibodies have been shown to substantially inhibit proliferation induced by LPS and F(ab')2 anti-Ig, and polyphosphoinositide hydrolysis induced by F(ab')2 anti-Ig. Surprisingly, however, whole anti-Ig was unable to inhibit induction of transferrin receptor (TfR) expression by LPS or F(ab')2 anti-Ig. Indeed, whole anti-Ig on its own induced TfR as early as 4 h. TfR induction by F(ab')2 anti-Ig and by LPS was accompanied by an early increase in TfR mRNA, and was prevented by inhibitors of protein and RNA synthesis and therefore can be ascribed to a transcriptional mechanism. In contrast, whole anti-Ig induced TfR even in the presence of protein and RNA synthesis inhibitors. Little or no TfR mRNA was detectable after 4 or 16 h of exposure to whole anti-Ig, whereas increased TfR mRNA was evident after 4 h of F(ab')2 anti-Ig or LPS. Antibody to the Fc gamma R (2.4G2) restores the ability of whole anti-Ig to generate increased TfR expression via the transcriptional route. We conclude that whole anti-Ig induces TfR mostly by using preexisting TfR molecules through a mechanism different from the transcriptional mechanism triggered by F(ab')2 anti-Ig and LPS.

Transferrin receptor synthesis is an early event in B cell activation.

Purified, small resting mouse B cells appear to express low levels of transferrin receptor (TfR) as detected by flow cytometry. Moreover, when such cells are stimulated with LPS or F(ab')2 anti-mu, they show increased expression of TfR as early as 4 h after activation when the cells are at the boundary of G0 and G1 phase by cell cycle analysis. Cells treated with anti-mu increased TfR expression gradually, reaching a plateau after 46 h, whereas cells with LPS reached their plateau by 12 h. The kinetics of induction of increased TfR expression was similar for both small and large B cells. Inhibition of protein synthesis with anisomycin or inhibition of transcription with actinomycin-D blocked the increased expression of TfR at 4 h. Northern blot analysis showed a marked increase of TfR mRNA at 2 and 4 h with either anti-mu or LPS which was then followed by an apparent decline at 16 h. These findings together with other recent studies justify reevaluation of the generally accepted models placing TfR induction late in the G1 phase of the lymphocyte activation sequence. They support the concept that in the B lymphocyte the TfR gene is selectively transcribed earlier than previously thought, before the general increase in RNA synthesis.

The influence of lipopolysaccharide content on the apparent B cell stimulating activity of anti-mu preparations.

Anti-mu preparations differ greatly in their ability to stimulate mouse B cells to incorporate tritiated thymidine (TdR). We have found that these differences may be due in part to different levels of lipopolysaccharide (LPS) content. In this report we show that LPS concentrations as low as 0.025 ng/ml stimulate the proliferation of T-depleted (C57BL/6 X DBA/2)F1 (B6D2F1) spleen cells, provided that 5 X 10(-5) M 2-mercaptoethanol is also present. Each of six commercial anti-mu preparations tested for LPS content contained more than this amount. We describe a technique that uses polymyxin B-agarose to remove nanogram quantities of LPS from anti-mu preparations. In B6D2F1 B cells, LPS-depleted anti-mu preparations induced much more uniform tritiated thymidine incorporation than did non-depleted preparations; but there was little difference between the two preparations when tested on B cells from C3H/HeJ (LPS hyporesponsive) mice.

Membrane depolarization of human B cells follows stimulation by either anti-mu or B-cell growth factor, but only anti-mu causes cell volume changes.

Human peripheral blood B cells were separated from monocytes and T cells, depleted of null cells by an anti-Leu 9 rosetting technique, and fractionated on discontinuous Percoll gradients to yield a highly purified, small, dense B-cell population. These cells responded to F(ab')2 goat anti-mu at 10 and 100 micrograms/ml with membrane depolarization (measured by immunofluorescence with 3,3'-dipentyloxacarbocyanine dye) at 1 h, cell volume enlargement by 48 h, and modest thymidine incorporation by 72 h. They also responded to the 12-kd human B-cell growth factor of Maizel with membrane depolarization, but not with cell volume increase. F(ab')2 anti-mu and B-cell growth factor together induced greater depolarization than was seen with either alone, but there was no synergy. The cell volume increase seen with F(ab')2 anti-mu was not increased by B-cell growth factor. Comparison of data analysis methods showed that mean fluorescence intensity most readily detected significant depolarization. We conclude that in human B cells: (1) depolarization may be a "general response" to a variety of membrane stimuli, because F(ab')2 anti-mu and B-cell growth factor acting through different receptors both induce it, and (2) depolarization does not inevitably lead to cell volume increase.