In vitro production of IgE by human peripheral blood mononuclear cells. I. Rate of IgE biosynthesis

IgE protein and grass-specific IgE antibodies were detected in the supernatants of 7-day cultures of unstimulated and pokeweed mitogen (PWM) stimulated human blood mononuclear cells from non-atopic and grass pollen-sensitive individuals. Significant amounts of IgE protein were detected in culture supernatants of grass-sensitive individuals and, even at lower levels, in those of non-atopic subjects. In contrast, detectable amounts of grass-specific antibodies were found only in the culture supernatants of grass-sensitive subjects. The mean values of total and grass-specific IgE detected in the supernatants of unstimulated and PWM-stimulated cultures did not differ statistically. Time sequence studies showed that IgE concentrations, measured in the 7-day supernatants, were due to a continuous release from the cells of IgE quantities progressively decreasing up to days 7 or 8. Comparison of the IgE protein and IgE antibody found in the 7-day culture supernatants to those released from initial cell pellets by treatment with acid buffer or freezing and thawing, showed that the IgE detected in 7-day supernatants could result, in part, from the release of cytophilic IgE bound to basophil or other cell types and in part also from the release of preformed lymphocyte cytoplasmic IgE into the supernatant fluids during the course of culture. In most non-atopic subjects and in some grass-sensitive patients the preformed IgE accounted virtually for the total IgE detected in the 7-day culture supernatants. However, the increase of IgE above the levels measured in the initial cell pellets, which was found in most grass-sensitive subjects, clearly reflected newly synthesized IgE. Both cycloheximide and puromycin were capable of reducing significantly the IgE concentration in culture supernatants when it was greater than the amount found in the initial cell pellets. The treatment of cells with mitomycin C was also able to decrease significantly the amount of IgE released in the supernatant after day 3 of culture.     

The spontaneous apoptotic cell death of normal human lymphocytes in vitro: the release of, and immunoproliferative response to, nucleosomes in vitro

Nucleosomes released spontaneously in short-term culture from murine spleen cells have a significant immunoproliferative effect in vitro, including the stimulation of anti-DNA antibody responses. The present studies show that during short-term cultures, tonsil lymphoid cells also undergo spontaneous apoptosis revealed morphologically (electron microscopy) by the appearance of changes in nuclear chromatin, typical of apoptosis and similar to morphologic changes of apoptosis in cultured normal splenic lymphocytes. This process is followed by the release, in the greater than 30-kDa cell free supernatant fraction, of core histones (H2A, H2B, H3, H4) and low molecular weight DNA (approx 160 bp) constituents of nucleosomes. The greater than 30-kDa tonsil lymphocyte cell-free supernatant material containing the constituents of core nucleosomes, as well as the greater than 30-kDa supernatant fractions of tonsil cell lysates harvested at the same time, had a significant immunoproliferative effect on human or murine lymphocytes, increasing both DNA and immunoglobulin synthesis (protein A plaque-forming cells). Thus the release of immunoproliferative nucleosomes form dying human lymphoid cells provides an autocrine lymphocyte stimulatory network which may be important in immunoproliferative disorders and in normal cell turnover. Apoptosis in vivo may also provide a potential source for the circulating nucleosomal DNA identified in plasma in some systemic lupus erythematosus patients as well as contributing to increased polyclonal B lymphocyte stimulation and autoantibody responses in this disorder.     

Increased susceptibility to in vitro ultraviolet B radiation in fibroblasts and lymphocytes cultured from systemic lupus erythematosus patients

Sunlight is known to induce exacerbations of systemic lupus erythematosus (SLE) but its mechanism remains unclear. We have previously reported that ultraviolet A (UVA) exposure induces an increase in total DNA synthesis (DS) in vitro but a decrease in unscheduled DNA repair synthesis (UDRS) of splenocytes of murine SLE strains. In order to investigate whether similar observations are characteristic of human SLE, peripheral blood lymphocytes (PBL) and dermal fibroblast (DF) cultures of 20 patients and 15 matched controls were exposed in vitro to UVA or UVB at different doses. Thirteen (65%) SLE DF cultures exposed to UVB light (12-24 J/m2) showed an increase in DS compared to paired unirradiated cultures. In contrast, UVB-irradiated DF from normal individuals had no significant increase in DS following UVB irradiation. When SLE DF were exposed to higher doses of UVB (48-96 J/m2), 90% of cultures showed a decrease in DS compared to only 20% in the control group. All of the SLE DF cultures showed a decrease of their unscheduled DNA repair capacity following UVB (24-48 J/m2) irradiation whereas no UDRS was apparent in 74% of controls under the same conditions. Similar findings regarding UDRS were observed in SLE PBL cultures and were also confirmed by autoradiography. UVA exposure (0-3840 J/m2) had no effect on DS nor on UDRS in DF or PBL cultured from SLE and controls. The relevance of these in vitro findings to the in vivo pathogenesis of the disease is discussed.     

In vitro reactivity of human lymphocytes in chronic uraemia: analysis and interpretation

Altered cellular immunity complicating chronic uraemia includes lymphocytopenia, thymic atrophy, impaired allograft rejection and delayed hypersensitivity in skin tests, diminished appearances of lymphocytes on skin windows, and shortened in vitro survival of uraemic lymphocytes. Studies were undertaken to further characterize these lymphocyte defects.

Lymphocytes separated from peripheral blood of twenty-four uraemic patients were compared with those from fifty-one normal persons in their rates of RNA and DNA synthesis in both PHA-stimulated and unstimulated cultures, as determined by incorporation of radiolabelled precursors. Synthesis, expressed as absolute incorporation/10⁶ viable lymphocytes, was accelerated in the majority of both stimulated and unstimulated uraemic cultures. Serial studies over several months of twenty-five uraemic patients (fifteen maintained by haemodialysis, ten by renal allotransplantation), compared with nineteen normal controls, showed these differences to be consistent and persistent. While normal lymphocytes exhibited stability, uraemic cells fluctuated widely in their serial synthesis rates. In 25% of such cultures, unstimulated synthesis exceeded that induced by PHA. Increased synthesis without PHA, reflecting enhanced spontaneous blastogenesis, is compatible with decreased survival, numbers, and functional capacity of uraemic lymphocytes.

Reports by others of diminished PHA-responsiveness of uraemic lymphocytes are based upon whole-culture incorporation and/or expressed as ratios between stimulated and unstimulated cultures. Both shortened survival and accelerated spontaneous nucleic acid synthesis by uraemic lymphocytes cause such ratios to be misleadingly low. Such factors as cell numbers and viability at harvest, counting efficiency, and culture sterility are essential to avoid misinterpretations of such data based on incorporation of radiolabelled precursors to the nucleic acids.

     

Immunoregulation in glomerulonephritis, Henoch--Schonlein purpura and lupus nephritis.

Immunoregulation was examined in normal controls and in patients with immune complex glomerulonephritis and lupus nephritis (SLE) using OKT monoclonal anti-bodies against helper (OKT4) and suppressor (OKT8) T cell subsets. Functional studies assessed T cell control of in vitro immunoglobulin synthesis by cultured peripheral blood mononuclear cells (PBMC). IgG and IgA synthesis was measured in unstimulated, pokeweed mitogen (PWM) stimulated and PWM + concanavalin A (Con A) stimulated cultures. Patients with primary membranous nephropathy (MN) and mesangial IgA nephropathy (IgA GN) were found to have elevated T4/T8 ratios secondary to a deficiency of the T8+ subset. Patients with SLE had low T4/T8 ratios. B cell activation with high spontaneous immunoglobulin synthesis was present in cell cultures from patients with SLE, IgA GN and Henoch-Schonlein purpura (HSP). Defective Con A inducible suppression of in vitro immunoglobulin synthesis was found in SLE, HSP and to a lesser extent, primary MN. Functional Con A inducible suppressor defects correlated with elevated T4/T8 ratios only in patients with MN. All four disorders appear to share disturbances of cellular immune response with various degrees of defective immune suppression; however, it is not clear from these studies whether the defects are primary or secondary phenomena.     

The synthesis and structure of visna virus DNA.

The synthesis of proviral DNA of visna virus was measured at various intervals after inoculation of sheep cell cultures at multiplicities of 0.3, 1, and 10 PFU/cell. The DNA from the infected cells was fractionated by the Hirt procedure into low (Hirt supernatant) and high (Hirt precipitate) molecular weight DNAs, and each fraction was quantitated for infectivity by plaque assay using the calcium phosphate transfection technique of Graham and Van Der Eb (1973). The appearance of infectious DNA in the Hirt supernatant (low molecular weight viral DNA) was biphasic at all multiplicities, apparently reflecting two rounds of synthesis of this DNA. The amount of infectious DNA in the Hirt precipitate fraction increased with time, reaching maximum levels in all cultures at the time of peak virus production. Hirt supernatant DNA consisted predominantly of molecules of molecular weight 6 × 10⁶, which appeared to be present as double-stranded linear molecules. Infectious DNA in the Hirt precipitate, in contrast, had a molecular weight of 166 x 106, suggesting its association with cellular sequences. The network test strongly suggested that the viral sequences were covalently linked with or integrated into the cellular DNA.     

Influence of supernatants from polymorphonuclear leucocytes on blastogenesis of syngeneic and allogeneic murine splenocytes.

Supernatant was produced from activated peritoneal polymorphonuclear leucocyte-rich cell populations from different strains of mice. These supernatants were studied for their ability to modify spontaneous and mitogen-induced blastogenesis of syngeneic and allogeneic splenocytes. Our results indicate that polymorphonuclear leucocyte-rich cell cultures from two strains of mice, A/J and BALB/c, produced a supernatant that could enhance PHA-induced blastogenesis of syngeneic and allogeneic splenocytes. Cells from a third strain C57B1/6, did not produce an active supernatant. In general, the response by splenocytes from these three strains paralleled the production of active supernatant that we observed. The response to the active supernatant was dependent upon the mitogen stimulation of the splenocytes, the mitogen dilution and the supernatant activity. These functions are believed to be genetically determined.     

Micro-system for the primary immunization of baboon (papio cynocephalus) peripheral blood mononuclear cells with sheep erythrocytes in vitro

A micro-culture system for the stimulation of baboon peripheral blood mononuclear cells (PBMC) with sheep erythrocytes (SRBC) in vitro was established. PBMC cultures in 96-well microtiter plates were maintained in 300 μl of culture medium containing a murine mixed lymphocyte reaction (MLR) supernatant and SRBC. Cultures of 7.5 × 10⁵ PBMC and 6 × 10⁶ SRBC resulted in the highest anti-SRBC plaque-forming cell (PFC) response. It was also determined that the presence of 50 μl of murine MLR supernatant was required for optimal PFC generation and that the cultures required supplementation with nutrient cocktail 3 days post-immunization. Although PFC were detectable on days 4–8, the maximum expression of PFC occurred on day 7.     

Investigations of intrinsic abnormalities in DNA-specific B lymphocytes from autoimmune mice

In murine models of systemic lupus erythematosus and in many humans with SLE, antibodies against native DNA (dsDNA) are a major contributor to the pathogenesis of the disease. Loss of self-tolerance to the DNA antigen may be associated with B-cell defects or regulatory cell dysfunction. We have developed B-cell lines with specificity for the antigen DNA, from both the autoimmune BWF1 mouse strain and from the non-autoimmune BALB/c strain, to use in the investigation of inherent B-cell defects in autoimmunity. Six BWF1 cell lines and five BALB/c cell lines which are free of Thy1.2+ cells and esterase positive cells, and have between 35 and 89% rosetting with dsDNA-SRBC targets, have been propagated in vitro for 24-36 months. The cells are non-malignant, growth-factor dependent and have no antigen or mitogen in the growth medium. Lyt-1 positive cells are found in the cell lines, but Lyt-1 negative cells are also present. They respond to the antigen DNA-HRBC when EL-4 supernatant is present in culture, and the peak of the plaque-forming cell (PFC) response is the same for both strains. When cells from both strains are cultured with varying amounts of T-cell factors, there is no difference in spontaneous antibody-forming cell (AFC) formation or in response to anti-mu stimulation between BWF1 and BALB/c strains. BALB/c spleen cells do not respond to DNA-HRBC in this culture system, but BWF1 spleen cells, as well as cell line cells from both strains, respond to this antigen. T cells from non-responding BALB/c spleen and responding BWF1 spleen are able to suppress the immune response to DNA-HRBC of cell line B cells from both strains. Propagating B-cell lines in the presence of DNA for 2 weeks stimulates BWF1 cell line cells, but suppresses the response of BALB/c cell lines to antigen.     

Mechanisms of action of `Lymphocyte activating factor'' (LAF): I. Association of lymphocyte activating factor action with early DNA synthesis in PHA-stimulated lymphocytes

The kinetics of the potentiation of phytohaemagglutinin-induced DNA synthesis by rat lymph node cells in the presence of non-mitogenic lymphocyte activating factor produced by rat peritoneal macrophages in serum-free cultures was studied. LAF, a dialysed 24 h supernatant of partially purified macrophages, caused a maximal potentiating effect when added to lymph node cell cultures between 0 and 24 h after their initiation. Addition at later times had a smaller enhancing effect. Removal of LAF from cultures after 16 h was too late to prevent a maximal LAF effect. Thus, optimal sensitivity to LAF was present at 16–24 h and decreasing sensitivity thereafter. This corresponds to the period of early DNA synthesis in the majority of phytohaemagglutinin-stimulated lymph node cells. Within this period, the minimal effective LAF exposure was 4.5 h and maximal potentiation was obtained with a LAF pulse of 6.5 h. The kinetic relationships observed were the same whether DNA synthesis was measured (by a [³H]-thymidine pulse) as early as 21–33 h after initiation of the cultures or as late as 48–65 h. The total effect, however, was greater when measured after 2 or 3 days of culture. Of interest, LAF also potentiated the responses of normal and ovalbumin-sensitized lymph node cells to phytohaemagglutinin and ovalbumin, respectively, in the presence of 2-mercaptoethanol. Therefore, 2-mercaptoethanol did not substitute for the biological activity of rat LAF as it is known to do with some macrophage functions.     

Multiple interactions between murine cytomegalovirus and lymphoid cells in vitro.

Spleen cultures from various strains of mice were infected in vitro with murine cytomegalovirus (MCMV). Infectious centres were established in a small proportion (not greater than 1%) of the cells. Virus could be rescued from these cells by co-cultivation with syngeneic or allogeneic fibroblasts, but the frequency of rescue could not be altered by incubation with cyclic nucleotide analogues, iododeoxyuridine, cortisol, or allogeneic spleen cells. In addition a smaller fraction of the cell population, possibly a sub-population of the infectious centres, replicated virus spontaneously. The presence of mitogens did not affect these interactions qualitatively or quantitatively. A third response to infection was an inhibition in DNA synthesis, which was suffered by unstimulated cultures and by cells transformed by concanavalin A and bacterial lipopolysaccharides, although overall cell viability was maintained. This response was also mediated by u.v.-inactivated virus.     

Immunization with the Sm nuclear antigen induces anti-Sm antibodies in normal and MRL mice.

The spontaneous occurrence of antibodies against the Sm nuclear antigen is a highly specific marker for the diagnosis of SLE. We have previously shown that anti-Sm can be elicited by immunization of SLE-prone mice with purified Sm antigen. In the present study, this autoantibody was induced in normal mice by a similar immunization protocol. Anti-Sm produced by normal strains was predominantly IgG1, which is similar to the isotype distribution in Sm-immunized MRL mice, but unlike the IgG2a-dominated response seen for spontaneous anti-Sm. Anti-Sm raised by immunization in most strains recognized epitopes not seen by spontaneous human and murine SLE anti-Sm; of the eleven normal strains tested, only C3H and AKR, strains from which MRL was partially derived, responded to these determinants. Further, immunoblot analysis of anti-Sm generated by immunization of MRL and normal mice revealed that the same proteins recognized by spontaneous human and murine anti-Sm were also seen by these sera. This study shows that an autoantibody highly characteristic of SLE can be produced in normal and MRL mice after appropriate immunization, and that the fine specificity of such experimentally induced antibody can be similar to that of spontaneous anti-Sm autoantibodies. The results imply a role for autoimmunization with Sm in the production of anti-Sm.     

Genetic basis of murine lupus

Systemic lupus erythematosus (SLE) is an autoimmune disorder characterized by the formation of a variety of autoantibodies and subsequent development of severe glomerulonephritis. Etiology of SLE remains unknown even if it is now well established that SLE is under polygenic control as well as the contribution of hormonal and environmental factors. The availability of several murine strains that spontaneously develop an autoimmune syndrome resembling human SLE, such as New Zealand, MRL and BXSB mice has provided useful tools for the genetic dissection of susceptibility to SLE. Moreover, development of various transgenic and mutant mice has made it possible to identify a number of susceptibility genes such as those involved in the regulation of apoptosis or B cell receptor signaling that can trigger lupus-like phenotypes. Obviously, further identification of the genetic defects present in lupus-prone mice is of paramount importance for understanding the immunopathogenesis of SLE.     

Effective phage therapy is associated with normalization of cytokine production by blood cell cultures

The aim of this study was to investigate the effect of phagotherapy on tumor necrosis factor alpha (TNF-alpha) and interleukin 6 (IL-6) serum levels and the ability of blood cells to produce these cytokines in culture. Fifty one patients with long-term, suppurative infections of various tissues and organs were enrolled. The ability of cells to secrete cytokines was tested using whole blood cell cultures, unstimulated or stimulated with lipopolysaccharide (LPS) from E. coli. In addition, cytokine serum levels were determined. Measurement of cytokine activity was performed using bioassays. We showed that TNF-alpha, but not IL-6 serum levels, were regulated upon division of patients into categories exhibiting initial: low, moderate and high cytokine levels. The low spontaneous production of IL-6 by blood cell cultures was elevated significantly on day 21 of phage therapy, whereas high release of this cytokine was inhibited. No such correlation was observed with LPS-induced IL-6 production in cell cultures when cells from low-, moderately- or highly-reactive patients were studied. Phage therapy modified TNF release according to the initial ability to produce that cytokine: it reduced TNF production in high responders and increased it in low responders. Patients infected only with Gram-positive bacteria demonstrated analogous changes in the spontaneous and LPS-induced TNF-alpha production as in the whole studied group. A similar kind of regulation was observed in TNF-alpha and LPS-induced production, i.e. low production was significantly elevated, high strongly inhibited, and moderate only slightly affected. In summary, we demonstrated for the first time that effective phage therapy can normalize TNF-alpha serum levels and the production of TNF-alpha and IL-6 by blood cell cultures.     

Enhancement of IgE synthesis and histamine release by T cell factors derived from atopic patients with bronchial asthma

Culture supernatants of unstimulated T cells (TCS) derived from normal donors or from atopic patients with bronchial asthma were tested for their ability to regulate the spontaneous IgE synthesis by B cells of normal and atopic subjects. The same TCS were also tested for their influence on the histamine release from leukocytes of house dust mites-sensitive patients. Addition of TCS to B cell cultures from allergic donors induced a dose-dependent increase of the spontaneous IgE production without affecting the synthesis of IgG, IgM, and IgA. The potentiating activity of TCS was observed only in B cell cultures spontaneously producing IgE; TCS were still active on irradiated B cells. The maximal IgE-enhancing activity was observed when TCS were added at the onset of B cell cultures. The supernatants of T cells lysed at day 0 did not contain IgE-potentiating factors. The antigen-induced but not the spontaneous histamine release from leukocytes of house dust mite-sensitive patients was enhanced by pretreatment with TCS from allergic donors. The enhancing activities of TCS on IgE synthesis and on histamine release could be removed by absorption with IgE-Sepharose and subsequently recovered by elution with glycine buffer. The results indicate that T cells of patients with asthma spontaneously release IgE-binding factors capable of increasing both the spontaneous IgE synthesis by B cells and the antigen-induced histamine release.     

Mitogen induction of murine C-type viruses. III. Effect of culture conditions, age, and genotype.

Various parameters affecting the induction of endogenous C-type virus from mouse spleen cell cultures by lipopolysaccharide (LPS), a B-lymphocyte mitogen, have been examined. Virus induction and mitogenicity, as measured by stimulation of intracellular DNA synthesis, showed very similar dose-response patterns suggesting that they are linked phenomena. 5-Bromo-2′-deoxyuridine (BrdU) enhances the virus induction by LPS in a dose-dependent way with an optimum concentration of 5 μg/ml. When the cell concentration was varied, optimal conditions for virus release were found to be between 2 × 10⁶ and 5 × 10⁶ cells/ml. Age dependence of virus induction was examined by comparing the effects of LPS and BrdU in cultures from old and young BALB/c mice. Cells from old mice released more virus in response to LPS than those from young mice, but additional BrdU treatment caused no enhancement of virus release. The mitogenic effect of LPS was found to be very similar in cultures from old and young mice. Spleen cultures from several strains of mice showed differing response patterns. While BALB/c, C57BL/6, and AKR cultures all released virus in response to LPS, additional BrdU treatment enhanced virus release from BALB/c and C57BL/6 but not from AKR cells. Cultures of 129/J mice are stimulated to divide by LPS but they could not be induced to release virus by this drug alone or in combination with additional BrdU. These results suggest that in B lymphocytes stimulated by LPS expression of endogenous viral genes is a function of age as well as genotype unlike their mitogenic response to LPS.     

Cell cycle-dependent multiplication of avian adenoviruses in chicken embryo fibroblasts

Summary Propagation of CELO virus employing confluent monolayers of chicken embryo fibroblasts (CEF) yielded virus titers one to two logs lower than those from confluent chicken kidney (CK) cells. An enhancement of virus production in CEF as measured by plaque formation was obtained by infecting cultures in the growing non confluent state. Measurements of³H-thymidine incorporation revealed a positive correlation between the DNA synthesis of CEF cultures at the time of inoculation and the amount of progeny virus, whereas in the CK-CELO-system no such relation was observed.Requirement of replicative fibroblasts for CELO multiplication was also demonstrated by comparison of virus replication in synchronized stationary and serum stimulated CEF cells. In stationary CEF cells arrested in the G₁ phase of the cell replication cycle by serum deprivation and infected with CELO virus, no cytopathic effect could be observed, and only very low amounts of virus were produced. But 24 hours after release of these cells for growth by serum stimulation a logarithmic rate of virus multiplication and a complete CPE occurred. Infection of synchronized CEF cultures at different stages of the cell cycle revealed that CELO multiplication was correlated with the S phase of the infected cell.In synchronized CELO infected CEF cultures viral DNA synthesis started 12 to 14 hours after growth stimulation when cells were near the end of the S phase. In contrast, no viral DNA synthesis could be measured in growth arrested CELO infected CEF cells, when cellular DNA synthesis was low. Therefore not only production of infectious virus but also viral DNA synthesis is correlated with events during the S phase of the infected CEF cell.With 7 FiguresPresented in part at the Arbeitstagung der Deutschen Gesellschaft für Hygiene und Mikrobiologie, Mainz, September 27–29, 1976.     

A measurable reduction of s.r. Ca content follows spontaneous Ca release in rat ventricular myocytes

 The Ca content of the sarcoplasmic reticulum (s.r.) was measured in voltage-clamped rat ventricular myocytes from the integral of the Na-Ca exchange current evoked by applying caffeine to release the s.r. Ca content. Following spontaneous release of Ca from the s.r., the s.r. Ca content was decreased. The magnitude of this decrease was equal to that of the amount of calcium directly measured to have been pumped out of the cell during the spontaneous release. Following a spontaneous release, the s.r. Ca content recovered linearly. These results are shown to be consistent with the hypothesis that the frequency of spontaneous release is determined by the time taken for the cell and s.r. to reaccumulate the Ca²⁺ ions pumped out of the cell during spontaneous release. Received: 9 July 1997 / Accepted: 21 July 1997     

B lymphocyte activation in systemic lupus erythematosus: spontaneous production of IgG antibodies to DNA and environmental antigens in cultures of blood mononuclear cells.

IgG antibodies to DNA, influenza virus haemagglutinin (HA), adenovirus hexon (HX) and mannan from Candida albicans (MN) have been determined in supernatants from 2-day unstimulated cultures of peripheral blood mononuclear cells from SLE patients and controls. Mean values were much higher in the SLE group, with from 20% (MN) to 85% (DNA) of patients giving values above the normal range. Although a significant correlation was observed between anti-DNA and anti-HA production, anti-HX and anti-MN showed no such correlations. The specificity of the ELISA assays was demonstrated by inhibition tests. It is concluded that a selective form of polyclonal activation in SLE results in the production of antibodies to foreign as well as to self antigens.     

Estrogen Induces Different Responses in Dermal and Lung Fibroblasts

Fibroblast-like cells derived from dorsal skin and lung of AKR strain mouse embryos were cultured with estrogen for 4 days. The labeled hydroxyproline content was measured as newly synthesized collagenous protein in the cell layer and media. Although collagen synthesis by both cell lines was increased in both fractions under the influence of β-estradiol-3-benzoate, at physiological concentrations of 10^-3-10^-1μ g/ml, the rate of increase differed. Fibroblasts derived from skin showed increased collagen synthesis of approximately 76%, while those from lung showed an increase of approximately 25%. Total protein synthesis by both cell lines also increased. In lung fibroblast cultures the synthesis of total protein was increased more than the synthesis of collagen; on the other hand in skin fibroblast cultures the synthesis of collagen was increased more than the synthesis of total protein. DNA synthesis by both cell lines was not affected by estrogen at the concentrations used. These findings suggest that fibroblasts develop in a different manner in each organ.