Influence of MHC background on the antibody response to detergent enzymes in the mouse intranasal test.

The mouse intranasal test (MINT) was developed to assess the immunogenic potential of detergent enzymes. The BDF1 mouse (H-2(b/d)), a cross of C57Bl/6 (H-2(b)) x DBA/2 (H-2(d)) has been used for most of the development work. Preliminary data in the CB6F1(H-2(d/b)), a cross of Balb/c (H-2(d)) x C57Bl/6 showed that this strain was similar to the BDF1 in its response to enzymes. These data also showed that the parental strains responded differently to the enzymes. To understand better the influence the major histocompatibility complex (MHC) background has on immune responses to enzymes, 3 different enzymes were tested in 4 inbred strains (C57Bl/6, DBA/2, Balb/c, and C57Bl/10), 2 hybrid strains (BDF1 and CB6F1), and 2 congenic strains (Balb.B10 and B10.D2). BDF1 mice rank enzymes the same as the guinea pig, which in turn correlates with sensitization in occupationally exposed humans. The ranking is based upon the dose of enzyme needed to give one-half maximal IgG1 antibody response (ED(50)) where Termamyl is more potent than Alcalase, which is equipotent to Savinase. The H-2(d) strains ranked the enzymes the same as the BDF1 but generated ED(50)s for the proteases that were one order of magnitude greater than the BDF1 ED(50)s. The response to Termamyl was the same in the two F1 strains and the H-2(d) strains. The H-2(b) strains did not rank the enzymes the same as BDF1 but the ED(50)s for the proteases were similar to the ED(50)s in the F1 strains. The response to Termamyl in the H-2(b) strains was lower than the response in the F1 and H-2(d) strains. Initial data show that the inbred strains will make enzyme-specific IgE antibody to high doses of enzyme with DBA/2 > Balb/c > C57Bl/6 in terms of the robustness of the response. The IgG1 responses in the congenic strains were similar to the responses in the H-2 matched strains. In addition, the antibody response to enzymes was consistent regardless of the source of BDF1 mice. The responses to these enzymes are clearly MHC linked with a role for Class II I-E molecules indicated. The current data strongly support the use of the F1 hybrid as an appropriate strain for evaluating allergic responses to enzymes.

Toxicology of protein allergenicity: prediction and characterization.

The ability of exogenous proteins to cause respiratory and gastrointestinal allergy, and sometimes systemic anaphylactic reactions, is well known. What is not clear however, are the properties that confer on proteins the ability to induce allergic sensitization. With an expansion in the use of enzymes for industrial applications and consumer products, and a substantial and growing investment in the development of transgenic crop plants that express novel proteins introduced from other sources, the issue of protein allergenicity has assumed considerable toxicological significance. There is a need now for methods that will allow the accurate identification and characterization of potential protein allergens and for estimation of relative potency as a first step towards risk assessment. To address some of these issues, and to review progress that has been made in the toxicological investigation of respiratory and gastrointestinal allergy induced by proteins, a workshop, entitled the Toxicology of Protein Allergenicity: Prediction and Characterization, was convened at the 37th Annual Conference of the Society of Toxicology in Seattle, Washington (1998). The subject of protein allergenicity is considered here in the context of presentations made at that workshop.

Immunologic cross-reactivity between respiratory chemical sensitizers: reactive dyes and cyanuric chloride.

BACKGROUND: CyCl is a low molecular weight reactive chemical used as an intermediate in the production of plastics, herbicides, pharmaceuticals, and fiber-reactive dyes. It is a potent inducer of specific IgE antibody. The CyCl functionality is a structural component of monochlorotriazine and dichlorotriazine dyes. OBJECTIVE: We have investigated the immunologic cross-reactivity between cyanuric chloride (CyCl) and reactive dyes and it was hypothesized that this moiety might be a dye epitope and that it might stimulate an allergic antibody response in dye-exposed workers. METHODS: To test this hypothesis, we have used sera with IgE antibodies to CyCl and also sera from dye-exposed workers who have IgE antibodies to Procion Orange MX2R, an azo dye containing the dichlorotriazine group. As a control group we have used dye-exposed workers with IgE antibody to Remazol Black B, a diazo dye containing the vinyl sulfone-reactive group. RESULTS: Using RAST and RAST inhibitions, we identified negligible cross-reactivity between CyCl and dichlorotriazine dye. CONCLUSION: The results of this study imply that the allergenic moiety on the dye residue resides in the chromophore rather than in the common structural component of CyCl and dichlorotriazine dyes.

Use of the mouse intranasal test (MINT) to determine the allergenic potency of detergent enzymes: comparison to the guinea pig intratracheal (GPIT) test.

A mouse intranasal test (MINT) was developed to determine the relative allergenicity of detergent enzymes. In this simple method, various doses of the enzymes are administered in a detergent matrix, via intranasal instillation, on days 1, 3, and 10, with serum samples collected on day 15. The sera are then analyzed for enzyme specific IgG1 antibody by an antigen specific enzyme immunoassay. The protease enzyme Alcalase (protease Subtilisin Carlsberg) has been used as a benchmark enzyme for development and characterization of the model. The objective of the current studies was to obtain potency comparisons with other protease and nonprotease enzymes and to begin to assess the validity of the model by comparison with potency determinations obtained with the guinea pig intratracheal (GPIT) test. The range of potencies detected among several enzymes of different classes was approximately 60-fold (compared with Alcalase). Modification of the dosing regimen to permit slightly more extended dosing did not change the relative potency determination. Comparison of data from the MINT and GPIT methods indicate that both the mouse and the guinea pig recognize the bacterial amylase Termamyl and a fungal exocellulase as more potent than Alcalase, a serine protease (Subtilisin B) and a fungal alpha-amylase (Fungamyl) as less potent than Alcalase, and the serine protease, Savinase, as equivalent to Alcalase. Also, these data are in alignment with clinical data on the prevalence of occupational enzyme sensitization. Given the simplicity and low cost of the MINT method compared with the GPIT test, these results support continued development of the method as an alternative approach for assessing the allergenicity of enzymes.

Safety assessment of enzyme-containing personal cleansing products: exposure characterization and development of IgE antibody to enzymes after a 6-month use test.

BACKGROUND: Enzyme-containing personal cleansing products were being considered for the consumer market. Although enzymes have been marketed safely for many years as ingredients in laundry products, their use in a personal cleansing application represented a new type of exposure for consumers that was not supported by the historical safety data. An exposure assessment and additional safety data would be needed before marketing to ensure consumer safety. OBJECTIVE: The work in this paper was designed to evaluate the potential for inhalation exposure to the enzyme during use of this new product while showering. Then a clinical trial was conducted to determine whether or not the level, duration, and routes of exposure encountered during use of this product would induce a Type I sensitization response to the enzyme. METHODS: Exposure was assessed during normal showering activities by collecting air samples with both high volume and personal samplers and quantitating enzyme levels with an ELISA. To assess the potential for sensitization, panelists were asked to use a prototype protease-containing bar product for all personal cleansing tasks and to keep a use diary reporting any associated symptoms. Physical and dermatologic examinations and skin prick tests with enzyme were conducted before the test commenced and at 2-month intervals. RESULTS: Exposure assessment results showed that airborne enzyme levels were primarily dependent on the concentration of the enzyme in the personal cleansing product. Mean values for total airborne enzyme protein ranged from 5.7 to 11.8 ng/m3 when enzyme concentration, time of use, and measurement technique remained constant. After 6 months of at-home product use, four of 61 test subjects using the enzyme-containing bar had positive skin prick test responses when tested with the enzyme. The skin prick test data were supplemented with serologic analyses, which detected IgE specific for the protease enzyme. None of these subjects showed any clinical symptoms indicative of allergic reaction. CONCLUSION: The ability of enzymes to induce development of allergic antibodies in this study led to the conclusion that this prototype enzyme-containing personal cleansing bar would represent an inappropriate use of enzymes in a consumer product application. The likelihood of both induction of an immunologic response and subsequent elicitation of allergy symptoms in a small but significant fraction of the user population was high. This finding resulted in the decision to halt further development of this prototype.

Respiratory allergenicity of detergent enzymes in the guinea pig intratracheal test: association with sensitization of occupationally exposed individuals.

A guinea pig intratracheal test was used to set occupational operating guidelines for new enzyme proteins used in the detergent industry. In these studies, animals were intratracheally dosed with different levels of enzyme protein and sera from the animals were titered for allergic antibody to the enzyme. The amount of antibody produced to an enzyme was compared to the amount of antibody produced to the same protein doses of Alcalase, for which effective operating guidelines exist. These comparisons were used to determine if a new enzyme was more potent, less potent, or equivalent to Alcalase; operating guidelines were then established for the new enzyme. Termamyl was about 10-fold more potent than Alcalase and the protease subtilisin B was shown to be less potent. Another protease, Savinase, was shown to be equivalent in potency to Alcalase. The operating guidelines for Termamyl were adjusted lower, whereas the operating guidelines for the proteases were set the same as that of Alcalase. Under these conditions, we would predict that sensitizations to new enzymes would be comparable to or lower than the sensitizations to Alcalase. Prospective evaluation of skin prick test data of factory workers showed that sensitizations to Termamyl and Savinase were similar to sensitizations to Alcalase. The sensitizations to subtilisin B were lower than those to Alcalase. During this time period (7 years), only three respiratory incidents (rhinitis) were reported, demonstrating that employees with positive skin prick tests can continue to work. These comparisons indicate that the guinea pig intratracheal test is a good animal model for evaluating enzymes as respiratory allergens and that the data generated can be used to set operating guidelines for occupational allergens.

Proteolytic detergent enzymes enhance the allergic antibody responses of guinea pigs to nonproteolytic detergent enzymes in a mixture: implications for occupational exposure.

A guinea pig intratracheal test was developed to assess the respiratory allergenicity of enzymes used in the detergent industry. Information gained from this test was used in a process for setting operational exposure guidelines to protect worker health. Mixtures of enzyme proteins were given to guinea pigs once per week for 10 weeks to determine whether there were interactions among enzymes that affected the induction of antibody responses to each enzyme in the mixture. Passive cutaneous anaphylaxis antibody titers against each enzyme were measured in sera. Mixtures of two or three enzymes always consisted of a protease (Alcalase, Savinase; Novo Industri A/S) with an alpha-amylase (Termamyl; Novo Industri A/S), a lipase (Lipolase; Novo Industri A/S), or both. Control animals were exposed to single enzymes. The antibody titers to Termamyl and Lipolase were significantly greater in animals dosed with the protease-containing mixtures as compared with control animals dosed with a single enzyme. Antibody titers to the protease were unchanged in the presence of additional enzymes in the mixture. Complete inactivation of protease activity abrogated the enhanced antibody response to Lipolase. Inhalation exposure of guinea pigs to a mixture of Alcalase and Lipolase also resulted in higher antibody titers to Lipolase as compared with animals exposed by inhalation to Lipolase alone, showing that the enhanced response was not due to intratracheal delivery of antigen to the respiratory tract. These results show that proteolytic enzymes in a mixture enhance antibody responses to other enzymes. This should be considered when defining exposure guidelines for protease-containing enzyme mixtures.

Effect of influenza virus infection on ovalbumin-specific IgE responses to inhaled antigen in the rat.

Upper respiratory tract viral infections have been reported in clinical studies to serve as risk factors for allergic sensitization. In order to study the relationship linking influenza virus illnesses to development of allergy, murine models of allergen sensitization were previously employed. These models showed that lethal influenza viruses were able to trigger allergen-specific immunoglobulin E (IgE) production and to inhibit tolerance to repeated exposure to aerosolized allergen in the mouse. The disadvantage of these murine models consists in the utilization of virulant and lethal strains of influenza virus. A nonlethal rat-adapted influenza virus (RAIV) host resistance model has been developed in our laboratory. It was used to evaluate the effect of influenza virus infection on IgE responses to inhaled ovalbumin (OA) in the rat. The high IgE-responder Brown-Norway (BN) rat was chosen for further study after comparing the IgE response to OA in Fischer 344 (F344) and BN rats. On d 1, BN rats were sensitized by administration of 1 mg OA subcutaneously alone or together with aluminum hydroxide (200 mg) and Bordetella pertussis (15 x 10(9) killed bacilli per rat in 1 ml), or only received saline. Rats were either infected with RAIV or sham-infected on d 0 (24 h prior to sensitization) or on d 15, 17, or 57. Rats were exposed for 3 min to aerosolized OA (OA 3% in phosphate-buffered saline) every week, starting on d 18. Serum OA-specific IgE was evaluated by reverse enzyme-linked immunosorbent assay (ELISA) 3 d after each OA challenge. BN rats elicited a detectable OA-specific IgE response that decreased after repeated aerosol exposures. Influenza virus infection transiently increased the OA-specific IgE response when rats were immunized with OA alone and were infected 1 d prior to the first challenge and also when rats received only saline on d 1, were exposed each week to aerosolized OA, and were infected prior to the seventh challenge. These results, with data previously reported in mice, emphasize the importance of upper respiratory tract viral infection in increasing IgE responses to allergens and may be of importance in human disease.

Workshop overview. Identification of respiratory allergens.

A variety of chemicals and proteins can sensitize the respiratory tract. Among these are materials of industrial importance, including certain diisocyanates, acid anhydrides, reactive dyes, and enzymes. Currently, no widely accepted or well-validated methods for the prospective identification of respiratory allergens exist. Most progress has been made with guinea pig methods where sensitizing potential is measured usually by assessment of changes in pulmonary function induced following sensitization and challenge. However, these methods are often prohibitively expensive, particularly for screening purposes. A number of alternative approaches are under consideration and are described here. The nature of the health problems associated with occupational respiratory sensitization, chemical structure-activity analyses as a tool for detecting pulmonary allergens, approaches used to test for respiratory allergens in guinea pigs, and alternative approaches using mice are all discussed. Finally, regulatory issues and needs with respect to respiratory sensitization are outlined.

Induction of type I hypersensitivity in guinea pigs after inhalation of phthalic anhydride.

Guinea pigs were exposed through inhalation to phthalic anhydride (PA) dust at 0.5, 1.0, and 5.0 mg/m3, 3 hours/day for 5 consecutive days. Inhalation challenge with aerosolized phthalic anhydride-guinea pig serum albumin (PA-GPSA) conjugate elicited immediate-onset respiratory reactions in animals exposed to all three levels of dust. Inhalation challenge of a subgroup of animals with phthalic anhydride dust did not elicit an immediate response, as measured by changes in respiratory frequency and plethysmograph pressure. Serologic studies showed that these animals had allergic IgG1a antibody to PA-GPSA. There was a dose-dependent increase in specific IgG antibody activity, as measured by ELISA. Animals exposed to and challenged with 5.0 mg/m3 PA dust had significant numbers of hemorrhagic lung foci. Those animals with the greatest number of foci had high IgG antibody activity to PA, as measured by ELISA. This study showed that exposure to levels of PA dust as low as 0.5 mg/m3, below the current threshold limit value of 6.0 mg/m3, can sensitize animals to produce allergic antibody.

A proposed screen for evaluating low-molecular-weight chemicals as potential respiratory allergens.

Allergic asthma can result when reactive low-molecular-weight chemicals (LMWCs) haptenate carrier proteins to form immunogenic conjugates, which then induce specific allergic antibodies. As part of an overall assessment process for evaluating the allergenic potential of LMWCs, an in vitro test for detecting the covalent derivatization of proteins by LMWCs was developed. In the assay, globulin-free serum albumins were incubated with increasing concentrations of a given LMWC and the mixtures separated via reversed-phase high-performance liquid chromatography (HPLC). Derivatization was monitored by shifts in the retention time of native versus modified protein. Retention time shifts were seen for most haptens when incubated with human serum albumin at a 50:1 (hapten:protein) starting molar ratio. Some haptens changed the retention time of the protein at a 5:1 initial ratio. Almost all chemicals that non-covalently bind to proteins did not change the protein retention time, even when incubated at 1500:1 molar ratios. The screen correctly identified 12/14 known human allergenic haptens and 23/24 non-allergenic LMWCs. It cannot detect sensitizers which must be metabolized into reactive haptens. This screen can be incorporated into an overall risk assessment approach for evaluating chemicals as respiratory allergens.

Respiratory and immunological responses of guinea pigs to enzyme-containing detergents: a comparison of intratracheal and inhalation modes of exposure.

Guinea pigs were exposed once a week for 10 weeks by intratracheal exposure to solutions of 3, 1, 0.3, or 0.1 micrograms of the enzyme protein, Subtilisn Carlsberg (Alcalase), in 250 micrograms of a detergent base. Other groups of guinea pigs were exposed by inhalation (6 hr per day, 4 days a week) to 1 mg/m3 of the aerosolized detergent base containing either 3.5, 1.1, 0.3, or 0.1% Alcalase protein. Evaluations of gross respiratory responses immediately following each intratracheal exposure revealed a significant dose response in respiratory symptoms measurable after the fourth exposure and continuing throughout the study. In the inhalation experiment, during Weeks 4 through 10, animals were observed to have respiratory symptoms which were dependent upon both the dose of enzyme and on total exposure to the enzyme/detergent atmosphere. For both intratracheal and inhalation routes of exposure, the initial appearance of respiratory symptoms coincided with the first appearance of measurable serum allergic antibodies specific to Alcalase. The allergic antibody levels increased with time and dosage by both routes of exposure, and the antibody titers generated by the intratracheal administration of antigen were comparable to those generated by the inhalation route of exposure. These results indicate that the intratracheal technique is appropriate for the evaluation of the respiratory allergic response to a protein.

A tier approach for evaluating the respiratory allergenicity of low molecular weight chemicals.

A multi-level approach for evaluating low molecular weight chemicals as respiratory sensitizers is proposed. The approach involves four levels of testing that utilize both in vitro and in vivo methods. Tier 1 evaluates structure-activity information to determine if the chemical can covalently modify carrier molecules. It also includes a literature search to determine if the compound belongs to a family of chemicals that has been reported to induce hypersensitivity. Tier 2 tests the chemical's potential to haptenate carrier molecules (i.e., protein) under in vitro conditions. Positive results in Tiers 1 and 2 lead to testing in a guinea pig injection model to assess chemical immunogenicity (Tier 3). A positive result at this level leads to testing in a guinea pig inhalation model to address questions about relevant routes of chemical exposure and allergenicity (Tier 4). Tier 4 results are used in determining safe chemical exposure levels. We have evaluated three chemicals using this scheme: phthalic anhydride, reactive black b dye, and toluene diisocyanate. All three have reactive groups and haptenate protein in vitro. They induce a humoral immune response when injected into guinea pigs at equimolar concentrations, and they sensitize animals via inhalation exposure. The severity of the response (antibody titer and respiratory reactivity) can be used to rank-order the chemicals in terms of allergenic "potency." Our data indicate that this approach can detect chemical allergens and can be used to characterize them as moderate or strong respiratory sensitizers.

ELISA for human IgE antibody to subtilisin A (Alcalase): correlation with RAST and skin test results with occupationally exposed individuals.

An ELISA was developed to detect specific IgE antibody to the Bacillus subtilis-derived proteolytic detergent enzyme, subtilisin A (Alcalase), in sera from exposed detergent workers. Workers in the detergent industry are exposed via inhalation to low levels of the enzyme dust in the presence of detergent dust. Chemically inactivated Alcalase was used as the test antigen. Significant binding of IgE antibody to the immobilized enzyme was detected in the ELISA. The binding of allergic antibody to Alcalase was specifically inhibited in a dose-dependent manner by preincubating sera with 0.1 to 100 micrograms of inactivated Alcalase. Binding of IgE antibody to Alcalase could not be inhibited by two other inactivated bacterial proteases, Savinase and Esperase, derived from different Bacillus species. ELISA and skin test results demonstrated total agreement for 27 of 31 samples (87%), whereas RAST and skin test results demonstrated total agreement for only 24 of 31 samples (77%), indicating that the ELISA was more sensitive than the RAST. We conclude that the ELISA is a sensitive, fast, alternative to the RAST for detection of Alcalase-specific IgE antibody in detergent enzyme-exposed workers.

Regulation of the antibody response by the acute phase reactant: mouse serum amyloid P-component (SAP).

Serum amyloid P-component (SAP) is the major acute phase reactant (APR) of mice. Purified mouse SAP at 0.1 to 10.0 micrograms/ml selectively suppressed the secondary in vitro IgG antibody plaque-forming cell (PFC) response to the T-dependent antigen TNP-KLH but not to the T-independent antigens TNP-LPS and DNP-Lys-Ficoll. The suppression was antigen nonspecific. The mechanism of suppression occurred primarily through the activation of Lyt-1+, I-J+ suppressor-inducer cells, which in turn activated a Lyt-2+ suppressor T-cell population. The activity of preexisting, antigen-specific Lyt-2+ suppressor T cells was not influenced by SAP. The antigen-nonspecific suppressor T cells generated by SAP were sensitive to cyclophosphamide. Removal of SAP from the culture fluid with rabbit anti-Mo SAP antibody or agarose beads abrogated the suppression. Pentraxin proteins closely related to mouse SAP, such as human SAP and hamster female protein (FP), also displayed immunoregulatory activity of the antibody response by the same cellular mechanism. The results suggest that SAP regulates antibody responses by the activation of suppressor-inducer T cells and that the regulation of the antibody response during the acute stage of inflammation may occur via SAP.

Enhanced interleukin 1 (IL-1) production mediated by mouse serum amyloid P component.

Purified serum amyloid P component (SAP), the major acute-phase reactant of mice, induces enhanced interleukin 1 (IL-1) production by elicited monocytes/macrophages in vitro. SAP also enhanced IL-1 elaboration by macrophages from lipopolysaccharide (LPS)-low responder mice and in the presence of polymyxin B, indicating that the small amounts of LPS present in the SAP preparation did not augment IL-1 production. Concentrations of SAP of 0.1 to 10.0 micrograms/ml enhanced IL-1 production by elicited and bacillus Calmette-Guerin (BCG)-activated peritoneal macrophages, but not by resident peritoneal macrophages. The inflammation-induced monocyte/macrophage population displayed selective binding of SAP. The mouse macrophage line P388D1, also could bind SAP and display enhanced IL-1 production in response to SAP. SAP did not bind to the macrophage cell line RAW264.7 nor did it enhance IL-1 secretion by this line. The results suggest that this acute-phase reactant has the potential to enhance inflammatory and immunological events mediated by IL-1.

Leukocyte adherence inhibition response to murine sarcoma virus-induced tumors. II. Requirement for T-cell subpopulations and macrophages.

Murine sarcoma virus (MSV)-immune T cells from C57BL/6 mice respond to intact RBL-5 tumor cells with the production of leukocyte adherence inhibition factor (LAIF), which mediates an adherence inhibition response of macrophages. LAIF is elaborated by isolated Lyt-2+ cells incubated with RBL-5 cells, whereas Lyt-1+ cells elaborate a substance that enhances macrophage adherence. Spleen macrophages or peritoneal exudate macrophages from MSV-immune mice when present at concentrations of 0.1% changed the response of Lyt-1+ cells from the formation of an adherence enhancing factor to the formation of an adherence inhibiting factor. Migration inhibition factor (MIF) was formed by Lyt-1+ cells, but not by Lyt-2+ cells under identical culture conditions. Addition of either spleen macrophages from mice with progressively growing tumors or tumor-infiltrating macrophages suppressed LAIF formation by both Lyt-1+ and Lyt-2+ cells. Tumor-infiltrating macrophages elicited an adherence enhancing factor from Lyt-2+ cells when present at high concentrations. The results suggest that the extent of macrophage adherence in vitro is the outcome of an interaction of macrophages with mediators that have opposing effects.